The Chemistry of Phytoplankton

Abstract

Phytoplankton have a high potential for CO₂ capture and conversion. Besides being a vital food source at the base of oceanic and freshwater food webs, microalgae provide a critical platform for producing chemicals and consumer products. Enhanced nutrient levels, elevated CO₂, and rising temperatures increase the frequency of algal blooms, which often have negative effects such as fish mortalities, loss of flora and fauna, and the production of algal toxins. Harmful algal blooms (HABs) produce toxins that pose major challenges to water quality, ecosystem function, human health, tourism, and the food web. These toxins have complex chemical structures and possess a wide range of biological properties with potential applications as new therapeutics. This review presents a balanced and comprehensive assessment of the roles of algal blooms in generating fixed carbon for the food chain, sequestering carbon, and their unique secondary metabolites. The structural complexity of these metabolites has had an unprecedented impact on structure elucidation technologies and total synthesis, which are highlighted throughout this review. In addition, the influence of biogeochemical environmental perturbations on algal blooms and their influence on biospheric environments is discussed. Lastly, we summarize work on management strategies and technologies for the control and treatment of HABs.

1. Introduction

During the first half of the 4.6 billion-year history of planet Earth, the atmosphere was devoid of free oxygen. Geological evidence indicates that cyanobacterial mats growing on stromatolite formations were the first oxygenic photoautotrophs responsible for harnessing solar energy by splitting water into hydrogen and oxygen over ∼ 3.5 billion years ago.⁹ This process fundamentally redefined the chemical environment of the biosphere of Earth 2.4 billion years ago (Figure 1), predating terrestrial plants by some 400 million years, and ultimately setting the stage for the Great Oxygenation Event (GOE) that is responsible for the life-enabling atmosphere that exists today. Now, cyanobacteria are globally distributed in nearly every euphotic environment, from remote ocean surface currents to urban stormwater ponds. The term phytoplankton refers not just to cyanobacteria but to a diverse set of microscopic photosynthesizers.¹ These include unicellular algae, such as cyanobacteria, diatoms, coccolithophores, and dinoflagellates.² These solar-powered biochemical reactors not only create the very environmental conditions that allow complex life to exist, but they also provide the base energy for countless food webs, drive biogeochemical cycles and facilitate diverse ecological interactions.

Figure 1.

Venus, Earth, and Mars represent the three rocky inner planets in our Solar System. The dramatic transformation of Earth’s atmosphere to a highly oxygenated system was initiated and sustained by phytoplankton, thus exemplifying the tremendous impact they have on the environment. (Images sourced from NASA).

The relatively high growth rate and high photosynthetic efficiencies of algal species, as compared to terrestrial plants, position algae as some of the most effective biological systems for atmospheric pCO₂ fixation and sequestration on the planet.^3,4 Phytoplankton are responsible for nearly half of the total global CO₂ fixation and form the base of the marine food web.⁵ They are often referred to as “blue carbon warriors” or “biological carbon pumps” for their roles against climate change by sequestering more than 50% of atmospheric pCO₂.⁶ Phytoplankton are responsible for mitigating the buildup of atmospheric pCO₂ via the biological pump and the production of calcium carbonate (CaCO₃),^7,8 processes that transport carbon to the deep ocean and sediments. The extensive evolutionary history of phytoplankton has resulted in the development of a range of ecophysiological adaptations and strategies.⁹ These adaptations ensure their survival and dominance in aquatic environments despite natural and human-induced negative environmental changes.^10,11

Today, anthropogenic nutrient enrichment (i.e., cultural eutrophication) and climate warming through increased atmospheric CO₂ make algal blooms occur more frequently and in remarkably broad geographic distributions. The changes in phytoplankton production and community composition are exemplified by the rise in harmful algal blooms (HABs), with around 150 occurrences of HABs recorded in 1987 worldwide to a total of nearly 10,000 cases from 1988 to 2023¹² (Figures 2a–c). Massive algal blooms are associated with various environmental drivers, including hypoxia, high pH, high temperature, dust storms, high concentrations of ammonia, potentially hypercapnia, and the loss of the benthic fauna and flora.^1000−15 It is extremely important to emphasize that some algal blooms produce toxins that are sequestered through bioaccumulation into food webs, causing the mortality of larger predators, such as marine mammals and sea birds, while also presenting health hazards for humans.⁶ In addition to toxin production, some phytoplankton species produce secondary metabolites that may inhibit (or stimulate) the growth of its microbial competitors in a process referred to as allelopathy.¹⁰⁰¹ Metabolomic studies have shown that allelopathic molecules from the same species can produce different modalities in target species ranging from highly inhibitory to sometimes even stimulatory.¹⁰⁰² For instance, the Florida red-tide producing dinoflagellate Karenia brevis, produces inhibitory metabolites with relatively higher concentrations of polyunsaturated fatty acid derived lipids or other aromatic compounds as well as stimulatory metabolites.¹⁰⁰² Future metabolomic investigations into allelochemicals will highlight the potentital chemical variability that exists in phytoplankton, especially when environmental drivers such as cultural eutrophication leads to unbalanced ratios of nitrogen and phosphorus.⁷⁰²

Figure 2.

a: Satellite images of large-scale phytoplankton blooms. a1: Algal bloom in Western Lake Erie on September 26, 2017. The Microcystis sp bloom shown is near downtown Toledo and stretches all the way to Lake Ontario. a2: Red tides in Benguela upwelling in 2008. a3: Cyanobacteria bloom in Lake Erie. (Photo courtesy of Tim Davis). a4: Algal bloom of Emiliania huxleyi off the southern coast of Devon and Cornwall in England in 1999. b: The taxonomic composition of HABs species (b1: Prymnesiophytes; b2: Diatoms; b3: Cyanobacteria; b4: Dinoflagellates). c: Total number of recorded HAB events (Y-axis) per year (X-axis) around the globe showing a steady upward trend.

Global climate data suggest that HABs will not only occur more frequently and widely but will also result in increased toxin production.^6,16−18 Despite the clear danger of HABs, there are some opportunities for fundamental research due to the potent activities and unique chemical structures of HAB toxins, which can be used to explore basic biological mechanisms and novel biosynthetic pathways.¹⁹⁻²¹ This review discusses what is known about the structural complexity and diversity of the chemistry of phytoplankton, highlights the CO₂ sequestration capacity of algal biomass, and discusses the occurrence and distribution of HABs. The bulk of the literature on this topic has been published between the 1980s to present, with a few reports dating between the 1950s–1970s.

2. CO₂ Sequestration and CaCO₃ Production

The major source of global CO₂ emissions is due to human activities. Anthropogenic fossil fuel combustion accounts for 89% of emissions, while the remainder stems from cement production and land use change, primarily deforestation. During the beginning of the Industrial Era in 1750s, the atmospheric pCO₂ concentration was approximately 278 parts per million (ppm) and has since risen to 417.1 ± 0.1 ppm in 2023.²² The heightened use of fossil fuels and biomass burning has led to a significant rise in greenhouse gases, with CO₂ constituting 76% of this increase.²³ The elevated atmospheric pCO₂ promotes dissolved inorganic carbon for heterotrophs (e.g., seagrasses) (Figure 3) especially in freshwater or brackish ecosystems that are not buffered by carbonate alkalinity.²⁴

Figure 3.

CO₂ emission and absorption.

Microalgae possess a unique system for CO₂ assimilation, referred to as the carbon concentration mechanism (CCM).⁴ They possess relatively high growth rates and a photosynthetic efficiency of 10–20% compared to 1–2% for terrestrial plants.²⁵ Despite constituting just 1–2% of the total carbon biomass of the world, phytoplankton in the oceans are responsible for fixing 45 to 50 billion metric tons of carbon each year. This accounts for nearly half of global CO₂ fixation.^1,5,26 Hence, phytoplankton are as important as the plant kingdom in regulating the carbon cycle of the Earth.²⁷ Interestingly, there is less than 1 gigaton (Gt) of phytoplankton alive in the ocean at any given time, but there is an estimated 45 Gt of new phytoplankton generated annually. This translates to the phytoplankton biomass cycling about 45 times per year. This high turnover of biomass is a major contributor to long-term carbon sequestration into the deep ocean and sediments.^28,29

Phytoplankton are responsible for the hydrogenous sequestration of CO₂ via the precipitation of CaCO₃ not induced by biological materials.³⁰ This is known as whiting events which largely occur in the Southwestern Atlantic Ocean, resulting in a nonskeletal carbonate factory driven by a Mediterranean outflow (Figure 4).³¹ For instance, the highly alkaline and saturated coastal waters of the Bahamas result in the formation of aragonite and calcite minerals, which are the two most common and stable forms of CaCO₃.³² Unlike most organic CaCO₃ sediment derived from macroscopic carbonate or skeletal grains, whiting events are a result of chemical reactions between alkaline minerals and CO₂ dissolved in relatively shallow and productive seawater.³² This production of CaCO₃ is believed to have a carbon negative role in the global carbon cycle by reducing the concentration of dissolved CO₂ in the ocean (pCO₂), thus combating climate change by reducing ocean acidification.³¹

Figure 4.

A Mediterranean outflow of highly alkaline water results in a nonskeletal carbonate factory that mitigates the effects of climate change by sequestering CO₂.

3. The Influence of Biogeochemical Environmental Perturbations on Algal Blooms

Biogeochemical environmental perturbations strongly impact the occurrence and intensity of algal blooms alongside the generation of HAB toxins in aquatic ecosystems³³ (Figure 5). Phytoplankton show efficient nutrient (nitrogen, phosphorus, iron, and trace metals) storage capabilities.³⁴ Some cyanobacteria species can utilize atmospheric nitrogen via N₂ fixation to support growth.^35,36 Numerous phytoplankton species (e.g., Trichodesmium spp.) are capable of altering their buoyancy to migrate, enabling them to access either the light-abundant conditions near the surface of the ocean or the nutrient-rich depths near the ocean’s nutricline (∼ 200 m).³⁷ Some phytoplankton species have formed mutualistic symbioses (e.g., between cyanobacteria and diatoms) to provide protection and nutrition cycling in nutrient-depleted waters.^38,39 Over the past several centuries, agricultural and industrial development has resulted in eutrophication, which leads to periodic proliferation and dominance of HABs.^10,11

Figure 5.

Summary of major approaches for mitigating HABs. Reduction of nutrient and greenhouse gas input is the most effective way to prevent HABs, but it is difficult to implement. The physical control of clay application to HABs has been widely used for a long time, but it is not suitable for large bodies of water. Biological controls, including parasites and viruses infecting dinoflagellates, are limited and can have consequences on the surrounding land.

Global warming caused by climate change and accumulated greenhouse gases can promote phytoplankton growth.⁴⁰ The decrease of pH in aquatic systems (i.e., ocean acidification) associated with greater dissolved CO₂ also may increase HAB growth. HAB growth activities can remain high even when the temperature exceeds 25 °C.³⁴ This enables HABs to be even more successful when competing with other eukaryotic primary producers since their growth rates begin to decline at higher temperatures. Reductions in Arctic Ocean ice cover during the summer has resulted in expansions of HAB events.¹⁰⁰³ The expansion of HAB events is likely to continue as these HAB species are mixotrophic and can survive by ingesting/grazing on bacteria as well as photosynthesizing.²⁴

The intensity and duration of precipitation and drought events associated with climate change-induced marine heat waves may also affect HAB growth. Freshwater may prevent blooms since it dilutes nutrient concentrations. However, larger and more intense precipitation events move nutrients from the land (i.e., runoff), which increases the amount of nutrients introduced into the aquatic system, which in turn promotes bloom growth.⁴¹ High concentrations of free nitrogen in drought waters can further promote N₂-fixing cyanobacterial blooms.⁴²

Some phytoplankton species produce prodigious amounts of dimethylsulfoniopropionate (DMSP) which is microbially converted to dimethylsulfide (DMS).¹⁰⁰⁴ DMS is volatile and can escape from the oceans to the atmosphere, where it oxidizes to cloud condensation nuclei (CCN). DMS oxidized CCN aerosols can significantly affect atmospheric chemistry and climatic processes such as the Earth’s radiation budget (Figure 3). The DMS cycle includes feedback loops with plankton communities that produce DMS.¹⁰⁰⁵ The DMS biogeochemical cycle is also intimately connected to the cycles of other important elements, especially C, N and Fe. In fact, DMSP production in the ocean is so significant that perhaps 3-10% of the primary production flows through DMSP in some regions.¹⁰⁰⁶ At the cellular level, DMSP can represent up to 60% of the cellular sulfur and, even more significantly, up to 16% of the cellular C content, of certain phytoplankton species.¹⁰⁰⁷ Thus, in addition to its importance in the sulfur cycle, the DMS cycle has much broader significance in ocean ecology and biogeochemistry.

4. HAB Toxins

Of the approximately 10,000 species of marine phytoplankton found in the oceans some 200 algal species have been identified as producing toxins, creating a major threat to human health, financial challenges, and ecosystem perturbations throughout the food web.⁴³ HAB toxins cover a broad spectrum of chemical classes from polyhydroxy polyenes,⁴⁴ alkaloids,⁴⁵ polyethers,⁴⁶ polypeptides,⁴⁷ and polyketides.⁴⁸ Based on the different clinical descriptions of associated illnesses these toxins are classified as amnesic shellfish poisoning (ASP), paralytic shellfish poisoning (PSP), diarrhetic shellfish poisoning (DSP), neurotoxic shellfish poisoning (NSP), and ciguatera poisoning (CP).⁴⁹ The poisonings are related to different toxins with distinct geographical distributions. ASP affects mainly the Atlantic and Pacific Canadian and US coasts, as well as the UK. PSP events occur more frequently in Canadian waters, the Atlantic US coasts, the Caribbean, South America, and the Philippines. DSP events have a much higher incidence in European seas and in the Mediterranean. NSP is confined to Florida, with a single outbreak reported from New Zealand. CP is mostly confined to the subtropical Pacific and the Caribbean, expanded to Micronesia, but also east and south Asia.^49,50 The most highly reported toxins are PSP and DSP (Figure 6).⁵⁰ HAB toxin chemical diversity and biological functions provide an entry point to understanding their effects on human health, pharmacology, and the environment.

Figure 6.

Geographical distributions of ASP, PSP, DSP, NSP and CP.

4.1. Polyhydroxypolyenes and Polyketides

4.1.1. Amphidinolides

The amphidinolides, first isolated in Okinawa, Japan, are a class of macrocyclic polyhydroxylated secondary metabolites isolated from the marine dinoflagellate Amphidinium sp.⁵¹ To date, amphidinolides A-Y (compounds 1–93) have been described.^20,51−74 With the exception of amphidinolides B₂ and B₃, which were isolated from Amphidinium sp. in Brewers Beach, St. Thomas, US Virgin Islands,⁵² all other amphidinolides were isolated from sources in Okinawa, Japan.

The structure of amphidinolide A has been proposed by Kobayashi (1991) based on nuclear Overhauser effect (NOE) experiments.⁷⁵ The initially proposed relative configuration assignment⁵¹ of amphidinolide A (1) was later corrected through a total synthesis that also established the absolute configuration.^76,77 Amphidinolide A showed cytotoxic activity against murine lymphoma cells, L1210 (IC₅₀ = 2.4 μg/mL), and against murine leukemia cells, L5178Y (IC₅₀ = 3.9 μg/mL).⁵¹

The absolute configuration of amphidinolide B²⁰ (2, later renamed amphidinolide B₁⁷⁸) was established through comparison of the retention times of amphidinolide B degradation products with those obtained through targeted synthesis using high-performance liquid chromatography (HPLC) analysis.⁷⁹ Amphidinolide B showed cytotoxicity against human colon tumor cell line HCT 116 with IC₅₀ value of 0.122 μg/mL.⁵² The total synthesis of amphidinolide B and amphidinolide B₂ (3) has been described.⁷⁸ Shimizu (1994) proposed the absolute configuration of amphidinolides B₂ and B₃ (4) on the basis of NMR spectra comparisons of closely related molecules whose structures were assigned using X-ray diffraction data.⁵² Amphidinolides B₂ and B₃ exhibited cytotoxicity against HCT 116 (7.5 and 0.206 μg/mL, respectively).⁵² Amphidinolides B₄ (5) and B₅ (6) (isolated from stain Y-100) exhibited potent cytotoxicity against L1210 (IC₅₀ = 0.00012 and 0.0014 μg/mL, respectively) and human oral epidermoid carcinoma KB cells (IC₅₀ = 0.001 and 0.004 μg/mL, respectively).⁵³ Amphidinolides B₆ (7) and B₇ (8) exhibited cytotoxicity against human B lymphocyte DG-75 cells (IC₅₀ = 0.02 and 0.4 μg/mL, respectively).⁵⁴ The relative and absolute configurations for amphidinolides B₄, B₅, B₆, and B₇ were assigned by the comparison of NMR and electronic circular dichroism (ECD) data with those of known amphidinolides.^53,54

An initial partial structural elucidation of amphidinolide C (9) led to the assignment of the relative configuration which was achieved through the combination of NMR data analyses and the synthesis of the segment.^55,80,81 Amphidinolide C exhibited potent cytotoxic activity against L1210 with an IC₅₀ value of 5.8 ng/mL.⁵⁵ The total synthesis of amphidinolide C has been reported.⁸²

Amphidinolide D (10) is a C-21-stereoisomer of amphidinolide B (55) and exhibited potent cytotoxicity against L1210 with an IC₅₀ value of 19 ng/mL.⁵⁶

Amphidinolide E (11) has cytotoxic activity against L1210 (IC₅₀ = 2.0 μg/mL) and L5178Y (IC₅₀ = 4.8 μg/mL) cells.⁵⁷ The absolute configuration was established through ECD data, the modified version of Mosher’s method, and oxidative degradation.⁸³ The structure was validated through total synthesis.⁸⁴

Amphidinolide F (12) has a configuration that matches amphidinolide C as evidenced by the high degree of similarity between their NMR spectra.⁵⁸ Amphidinolide F exhibited cytotoxic activity against L1210 cells and KB cells with IC₅₀ values of 1.5 and 3.2 μg/mL, respectively.⁵⁸ The total synthesis of amphidinolide F has been reported.^82,85,86

Amphidinolides G (13) and H (14) (both isolated from strain no. Y-72) exhibited extremely strong cytotoxic activity against L1210 with IC₅₀ values of 5.4 and 0.48 ng/mL and KB with IC₅₀ values of 5.9 and 0.52 ng/mL, respectively.⁵⁹ The absolute configurations of amphidinolides G and H were determined by X-ray diffraction data analysis, synthesis of a degradation product of amphidinolide H, and interconversion between amphidinolides G and H.⁸⁷ The total syntheses of amphidinolide G and H have been reported.⁸⁸ Amphidinolides G₂ (15), G₃ (16), H₂ (17), H₃ (18), H₄ (19), and H₅ (20) (all isolated from strain no. Y-42) exhibited cytotoxic activities against L1210 with the IC₅₀ values of 0.3, 0.72, 0.06, 0.002, 0.18, and 0.2 μg/mL and KB with IC₅₀ values of 0.8, 1.3, 0.06, 0.022, 0.23, and 0.6 μg/mL, respectively.⁶⁰ The relative configurations were determined by NOESY data and by comparison of the NMR spectroscopic data of amphidinolides G and H.⁶⁰ The absolute configuration of amphidinolide H₂ was determined by chemical degradation and the Mosher method⁶⁰ and later revised by stereoselective synthesis.⁸⁹

Amphidinolide J (21) was cytotoxic against L1210 and KB (IC₅₀ = 2.7 and 3.9 μg/mL, respectively).⁶¹ The absolute configuration was established through the synthesis of its ozonolysis products.⁶¹

Amphidinolide K (22) showed cytotoxic activity with IC₅₀ values of 1.65 and 2.9 μg/mL against L1210 and KB cells, respectively.⁶² The absolute configuation was tentatively established by NMR experiments and later verified through total synthesis of natural amphidinolide K⁹⁰ and of its enantiomer.⁹¹

Amphidinolide L (23) showed cytoxicity against L1210 and KB with IC₅₀ values of 0.092 and 0.1 μg/mL, respectively.⁶³ The relative configuration was assigned by NOESY data and J-based configurational analysis (JBCA). The absolute configuration at C-22, C-23, and C-25 was determined through synthesis of a fragment containing these elements.⁶³

Amphidinolides M⁶⁴ (24) and N⁹² (25) exhibited cytotoxic activity against L1210 and KB cells with IC₅₀ values of 1.1 and 0.44 μg/mL and 0.05 and 0.06 ng/mL, respectively. The relative configuration of amphidinolide N was assigned by conformational analysis based on NMR experiments.^93,94

Amphidinolide O (26) and P (27) have cytotoxicity against L1210 (IC₅₀ = 1.7 and 1.6 μg/mL, respectively) and KB cells (IC₅₀: 3.6 and 5.8 μg/mL, respectively).^65,95 The absolute configurations of amphidinolides O (26) and P were assigned based on the specific rotations of the compounds prepared through an enantioselective synthesis.⁹⁶⁻⁹⁸ The total synthesis of amphidinolide P has been reported.⁹⁹

Amphidinolide Q (28) exhibited moderate cytotoxicity against L1210 cells (IC₅₀ = 6.4 μg/mL).⁶⁶ Its absolute configuration was determined through a modified Mosher’s method.¹⁰⁰ The enantioselective total synthesis has been accomplished.¹⁰¹

Amphidinolides R (29) and S (30, both isolated from strain no. Y-5) showed cytotoxicity against L1210 (IC₅₀ = 1.4 and 4.0 μg/mL) and KB cells (IC₅₀ = 0.67 and 6.5 μg/mL), respectively.⁶⁷ The absolute configurations of both amphidinolides R and S were determined based on chemical derivatization experiments and spectroscopic data.⁶⁷

Amphidinolides T (31, also named amphidinolide T₁)⁶⁸ and T₅⁶⁹ (32) were derived from Amphidinium sp. strain no. Y-56 while amphidinolides T2 (33), T3 (34), and T4 (35) were derived from Amphidinium sp. strain no. Y-71.¹⁰² The relative configuration of amphidinolide T was deduced from the NOESY correlations and the absolute configuration was assigned by Mosher’s method, degradation products,⁶⁸ and single crystal X-ray diffraction data analysis.⁶⁹ The absolute configuration of C-7, C-8, and C-10 of amphidinolides T₂–T₄ were determined by comparison of NMR data of their C-1-C-12 segments with those of synthetic model compounds for the tetrahydrofuranyl motif.¹⁰² The relative configuration of amphidinolide T₅ was solved through comparison of NMR data with amphidinolides T₃ and T₄; the absolute configuration of amphidinolide T₅ was confirmed with a modified Mosher’s method.⁶⁹ Amphidinolides T₁–T₅ exhibited cytotoxicity against L1210 cells (IC₅₀: 18, 15, 10, 7, and 11 μg/mL, respectively) and KB cells (IC₅₀: 35, 20, 11.5, 10, and 18 μg/mL respectively).^68,69,102 The total syntheses of amphidinolides T₁, T₃, T₄, and T₅ have been reported.¹⁰³

Amphidinolide U (36, isolated from strain no. Y-56) showed cytotoxicity against L1210 (IC₅₀ = 12 μg/mL) and KB cells (IC₅₀ = 20 μg/mL).⁷⁰ The relative configuration of C-15, C-18, and C-19 was deduced from NOESY correlations while the absolute configuration at C-8 and C-24 were assigned on the basis of a modified Mosher’s method.⁷⁰

Amphidinolide V (37, isolated from strain no. Y-56) exhibited cytotoxicity against L1210 (IC₅₀ = 3.2 μg/mL) and KB cells (IC₅₀ = 7 μg/mL).⁷¹ The absolute configuration of amphidinolide V was elucidated by enantioselective total synthesis.¹⁰⁴

Amphidinolide W (38, isolated from strain no. Y-42) exhibited cytotoxicity against L1210 cells with an IC₅₀ value of 3.9 μg/mL.⁷² The absolute configuration of amphidinolide W was initially assigned through the combination of JBCA and a modified Mosher’s method,⁷² and later revised after an enantioselective total synthesis was completed.¹⁰⁵

Amphidinolide X (39, isolated from strain number, Y-42) exhibited cytotoxicity against L1210 and KB cells with IC₅₀ values of 0.6 and 7.5 μg/mL, respectively.⁷³ The relative and absolute configuration of amphidinolide X were determined by combined analyses of NOESY, JBCA and NMR data of the degradation products. The total synthesis of amphidinolide X has been reported.¹⁰⁶

Amphidinolide Y (40, isolated from strain no. Y-42) is cytotoxic against L1210 and KB cells with IC₅₀ values of 0.8 and 8.0 μg/mL, respectively.⁷⁴ The relative configuration of amphidinolide Y was deduced by NOESY and JBCA analysis, and the absolute configuration was determined by Mosher’s method and ECD chiroptical data.⁷⁴ The total synthesis of amphidinolide Y has been reported.¹⁰⁶

4.1.2. Amphidinols

Amphidinols (AMs) are a group of polyhydroxy polyene compounds that were originally isolated from Amphidinium klebsii.^107−110 During the process of examining dinoflagellate cultures for bioactive substances, Masayuki (1991) stumbled upon a potent antifungal compound, known as AM, within cultures of the dinoflagellate A. klebsii.(107) Subsequently, from the surface wash of seaweeds collected at Aburatsubo Bay near the Miura Peninsula, Japan, Gopal (1995) isolated a strain of A. klebsii from which AM 2 was extracted.¹⁰⁸ In the following decades, additional AMs, such as AM 3 and AM 22, were successively isolated.^109,110 AM (41) was isolated from A. klebsii on the Ishigaki Island, Japan.¹⁰⁷ The configuration of AM remains unknown because its 27 stereogenic centers were remote, and most of them resided on acyclic residues.¹⁰⁷ AM exhibited potent antifungal (Aspergillus niger) growth-inhibiting activity = 6 μg/disk.¹⁰⁷ AM 2 (42) was isolated from A. klebsii from Aburatsubo Bay near the Miura Peninsula, Japan.¹⁰⁸ AM 2 showed potent hemolytic activity against human erythrocytes with a half-maximal effective concentration (EC₅₀) of 7.3 nM, which was about several hundred times more effective than the standard saponin. It also possesses antifungal activity (6 μg/disk) against A. niger.(108) AM 3 (43) was isolated from A. klebsii on Ishigaki Island, Japan.¹¹⁰ The absolute configuration of AM 3 was first established by JBCA¹¹⁰ and a modified Mosher’s method.¹¹¹ The structure was revised after GC-MS analysis of the degradation product¹¹² and stereoselective synthesis.¹¹³ AM 3 caused rapid hemolysis at 1.3 μM.⁴⁴ The total synthesis of AM 3 has been reported.¹¹⁴ AM 3 showed antifungal activity against A. niger with a minimal effective concentration (MEC) value of 9.0 μg/disk.⁴⁴ AM 5 (44) and AM 6 (45) were isolated from A. klebsii at Aburatsubo Bay.¹¹⁵ AM 5 and 6 showed anti-A. niger activity, hemolytic and antidiatom activities (AM 5:6 μg/disk, 0.23 μM, 0.5 μg/mL, respectively; AM 6:6 μg/disk, 0.58 μM, 1.0 μg/mL respectively.)¹¹⁵ AM 7 (46) was isolated from A. klebsii from Aburatsubo Bay.¹¹⁶ Based on analysis of the NMR data, it was concluded that AM 7 and AM 3 had the same relative configuration at C-11-C-52.¹¹⁶ AM 7 showed antifungal activity against A. niger with an MEC of 10 μg/disk and hemolytic activity with an EC₅₀ of 3 μM.¹¹⁶ AM 4 (47), 9 (48), 10 (49), 11 (50), 12 (51), and 13 (52) were isolated from Amphidinium carterae collected in New Zealand.¹¹⁷ The absolute configurations of AM 4 and AM 12 were assumed to be the same as AM 3.¹¹⁷ The hemolytic and antifungal activities against A. niger, and cytotoxicity against murine leukemia cell line P388 of AM 4 were 0.207 μM, 58.2 μg/disk, and 25.3 μg/mL.¹¹⁷ The hemolytic and antifungal activities against A. niger, and cytotoxicity against P388 cells of AM 9 were 0.176 μM, 32.9 μg/disk, and 36.5 μg/mL, respectively.¹¹⁷ Activities for AM 10 were 6.53 μM, 154.0 μg/disk, and 35.2 μg/mL, respectively,¹¹⁷ AM 11 were 28.9 μM, 256.6 μg/disk, and 23.0 μg/mL, respectively,¹¹⁷ AM 12 were 2.99 μM, > 100 μg/disk, and 26.8 μg/mL, respectively, and AM 13 were 2.02 μM, 132.0 μg/disk, and 32.5 μg/mL respectively.¹¹⁷ AM 14 (53) and 15 (54) were isolated from A. klebsii at the Aburatsubo Bay in the Miura Peninsula, Japan.¹¹⁸ The strong similarities between the NMR spectroscopic data suggested that the configuration of the shared segment between AM 14 and 15 should match that of AM 7.¹¹⁸ AM 14 exhibited no antifungal activity against A. niger.(118) AM 17 (55) was isolated from A. carterae in Little San Salvadore Island, Bahamas.¹¹⁹ AM 17 exhibited an EC₅₀ of 4.9 μM in a hemolytic assay using human red blood cells but displayed no detectable antifungal activity against A. niger (ATCC 16404) or Candida kefyr (ATCC 2512).¹¹⁹ AM 18 (56) and 19 (57) were isolated from A. carterae in Naples, Italy.¹²⁰ Portions of AM 18 and 19 were considered to have similar absolute configurations as AM 3 based on similarities between their NMR spectra.¹²⁰ AM 18 exhibited an antifungal activity against Candida albicans at 9 μg/mL.¹²⁰ AM 20 (58) and 21 (59) were isolated from A. carterae collected in Korea.¹²¹ The partial relative configurations of AM 20 and 21 were deduced from NOE data.¹²¹ The hemolytic and antifungal (A. niger) activities of AM 20 were 1–3 μM and >15 μg/disk, respectively.¹²¹ The hemolytic and antifungal (Aspergillus niger) activities of AM 21 were >10 μM and >15 μg/disk, respectively.¹²¹ AM 22 (60) was isolated from cultured A. carterae (CCMP1314).¹⁰⁹ The IC₅₀ values of AM 22 on to human non-small lung cancer cells A549, human melanoma A2058 cells, human hepatoma HepG2 cells, human breast adenocarcinoma cells MCF7, and pancreatic cancer cell line MiaPaCa-2 were 8, 16.4, 6.8, 16.8, and 8.6 μM, respectively.¹⁰⁹ AM 24 (61), 25 (62), and 26 (63) were isolated from A. carterae strain ACRN03 in Vigo, Spain.¹²² The partial relative configurations of AM 24-26 were established via NOE and rotating frame Overhauser effect (ROE) data.¹²² AM A (64) and B (65) were isolated from a strain of A. carterae of Lake Fusaro near Naples, Italy.¹²³ The partial relative configurations of AM A and B were established by J-coupling and NOE analysis.¹²³ AM A showed antifungal activity against C. albicans (MIC = 19 μg/mL).¹²³ AM C (66) was isolated from A. carterae in Ireland.¹²⁴ The proposed relative configuration of AM C was established based on a putative biosynthesis scheme.¹²⁴ AM C showed antifungal activity against Aspergillus flavus and C. albicans with minimal effective concentration (MIC) values of 4 and 16 μg/mL, respectively.¹²⁴

4.1.2.1. Biosynthesis of Amphidinol

Toshihiro (2001) proposed the acetate incorporation patterns of AM 2–4 (42, 43, 47, respectively).¹²⁵ The experiments revealed that they were constructed with five regular C2-elongation sequences.¹²⁵ These findings supported the C1-deletion mechanism from the regular sequence, which could be accounted for either by a Favorskii-type reaction or by a Tiffeneau-Demjanov-type rearrangement.¹²⁵ The first two carbons were not labeled with acetate but were subsequently shown to come from glycolate, a photorespiration product.¹²³ AMs are biosynthesized from the polyhydroxy end to the polyolefin terminus. The great variation in the structures of the polyhydroxy moiety might be attributable to the truncation of a polyketide chain during biosynthesis, as suggested for okadaic acid analogs.^116,125

4.1.2.2. Synthesis of Amphidinol 3 (AM3)

Amphidinol 3 (43) is a C₇₀ long chain polyol. It comprises of 25 stereogenic carbon atoms, two tetrahydropyran rings, and nine olefins. The complexity of the molecule, in combination with the low amount originally isolated, were among the contributing factors that led to the mischaracterization of AM3, which has since been revised three times, namely at C-2, C-32-C-36, C-38, and C-51, with the most recent revision being reported in 2018.^112,113,126 Efforts by Evans and Yadav report the preparation of AM3 fragments bearing the correct structural assignment.^127,128 The first total synthesis of AM3 was reported by Oishi in 2020, and confirmed the revised structure as correct.¹¹⁴ This synthesis provides AM3 in 112 steps and was accomplished through the late-stage combination of the C-1-C-29, C-30-C-52, and C-53-C-67 fragments of AM3. The longest linear sequence of this highly convergent synthesis is 40 steps.

The C-1-C-29 fragment was prepared using a Julia-Kocienski olefination (Scheme 1), and the synthesis started from compound 67 [(prepared in three steps from (R)-glycidol)].¹²⁹ Olefin metathesis with acrolein gave α, β-unsaturated aldehyde 68. Oxa-Michael addition mediated by Bi(NO₂)₃ followed by reduction of the aldehyde gave primary alcohol 69. Protection of the alcohol, desilylation, and oxidation gave aldehyde 70. Brown crotylation of 70 with 71 gave homoallylic alcohol 72 in 69% yield. Hydrolysis of the acetonide, global protection of the hydroxy groups as TBS-ethers, and hydroboration/oxidation of the olefinic moiety gave primary alcohol 73. Conversion of alcohol 73 to sulfone 74 in two steps afforded one Julia-Kocienski olefination partner for the C-1-C-29 fragment. The aldehyde coupling partner for the olefination was prepared from 75. The addition of a lithium acetylide to Weinreb amide 75 followed by asymmetric reduction of the ketone using Ru(II) catalyst 76, saturation of the alkyne, and silylation gave amide 77. Addition of the lithio anion derived from 78 to amide 77 provided ketone 79 quantitively. Borane reduction of the ketone with a Corey-Bakshi-Shibata (CBS) catalyst followed by silylation gave TBS-ether 80. Olefin metathesis of 80 with homoallylic alcohol 81 followed by silyation gave compound 82. Oxidative deprotection of the PMB-ether followed by oxidation of the resulting alcohol gave aldehyde 83 in 67% yield over the two steps. Julia-Kocienski olefination with 74 and 83 gave 84 in 86% yield. Sharpless dihydroxylation of the newly formed double bond was followed by silylation to the corresponding TBS-ethers. Removal of the 2-naphthylmethyl (NAP) protecting group followed by Grieco elimination gave the C-1-C-29 fragment 85.

Scheme 1. Synthesis of the C-1-C-29 Fragment.

a. acrolein, Hoveyda-Grubbs II, DCM, 72% yield; b. acetaldehyde, Bi(NO₃)₃·5H₂O, DCM; c. NaBH₄, MeOH, 87% yield (2 steps); d. NapBr, NaH, DMF, 82% yield; e. TBAF, THF, 96% yield; f. DMP, DCM, 89% yield; g. 71, THF, 69% yield; h. TsOH·H₂O, 89% yield BRSM; i. TBSOTf, 2,6-lutidine, quant.; j. BH₃·SMe₂, CyH; NaOH, H₂O₂, 91% yield; k. 1-phenyl-1H-tetrazole-5-thiol (PTSH); PPh₃, DIAD, 91% yield; l. m-CPBA, 86% yield; m. HC≡CC₄H₈OPMB, n-BuLi, 81% yield; n. Ru cat. 76, IPA, 90% yield, 94% ee; o. H₂, Pd/C(en), 89% yield; p. TBSCl, imidazole, DMF, quant.; q. 78, n-BuLi, THF, quant. r. (S)-(−)-2-methyl-CBS-oxazaborolidine, BH₃·SMe₂, PhMe quant. dr 95/5; s. TBSCl, imidazole, DMF, quant.; t. Hoveyda-Grubbs II, DCM, 70% yield, E/Z = 13/1; u. TBSOTf, 2,6-lutidine, quant.; v. DDQ, pH 7 buffer, DCM; w. SO₃·Py, DMSO, NEt₃, 67% yield (2 steps); x. 74, KHMDS, THF, 86% yield; y. K₂OsO₄·2H₂O, DHQ-MEQ, K₂CO₃, K₃Fe(CN)₆, MeSO₂NH₂, t-BuOH/t-BuOMe/H₂O, 65% yield, dr 13/1; z. TBSOTf, 2,6-lutidine, DCM, 93% yield; aa. DDQ, pH 7 buffer, DCM, then NaBH₄, MeOH/THF, 83% yield; ab. o-O₂NC₆H₄SeCN, PMe₃, 4 ÅMS, THF, then aq. H₂O₂, 97% yield.

The strategy for the synthesis of the C-30-C-52 fragment relied on the late-stage coupling of the A and B tetrahydropyran ring systems. The synthesis of this fragment is summarized in Scheme 2.¹¹³ Vinyl iodide 86 (prepared in five steps from ethyl propiolate) was subjected to olefin metathesis with ethyl acrylate in 72% yield. This new acrylate was reduced with DIBAL, and the alcohol was protected as a benzyl ether (compound 87). The Δ^5,6 double bond of 87 was subjected to dihydroxylation followed by acetylation to afford compound 88. Suzuki cross coupling of vinyl iodide 88 and vinyl Bpin (boronic acid pinacol ester) 89 gave diene 90 in 87% yield. Desilylation of 90 followed by alcohol-directed alkene epoxidation and acetate hydrolysis gave epoxide 91. Acid-catalyzed epoxide ring opening led to tetrahydropyran 92, which established the A ring. Acetonide protection of the 1,3-diol, dihydroxylation of the alkene with subsequent protection as the acetonide, and mesylation of the remaining hydroxy group gave tetrahydropyran 93. Hydrogenolysis of the benzyl ether followed by base initiated β-elimination led to epoxide 94 in good yields. Addition of lithio dianion 95 to epoxide 94, followed by silylation, produced terminal alkyne 96. Reductive iodination of alkyne 96 followed by exchange of the PMB-ether for a TES-ether gave vinyl iodide 97. Metal–halogen exchange of vinyl iodide 97 with t-BuLi at low temperature followed by addition to known aldehyde 98(113) gave a 2.2:1 mixture of separable C-43 epimers. With the core structure of AM3 prepared, the fragment was prepared for a cross coupling reaction with the C-1-C-29 fragment. Following silylation to 99, the TES ether was selectively cleaved, and the resulting alcohol oxidized to aldehyde 100. Homologation to the terminal alkyne via the Bestman-Ohira reagent was followed up by methylation to internal alkyne 101. Finally, the C-30-C-52 fragment precursor 101 was converted to vinyl iodide 102 through the corresponding vinyl stannane.

Scheme 2. Synthesis of the C-30-C-52 Fragment.

a. ethyl acrylate, Hoveyda-Grubbs II, 72% yield; b. DIBAL, DCM, 95% yield; c. NaH, BnBr, DMF, 87% yield; d. K₂OsO₄·2H₂O, DHQ-MEQ, K₂CO₃, K₃Fe(CN)₆, MeSO₂NH₂, t-BuOH/t-BuOMe/H₂O,, 83% yield, dr 7.5/1; e. Ac₂O, DMAP, pyridine, 93% yield; f. 89, PdCl₂(dppf), aq. Cs₂CO₃, DMF, 87% yield; g. HF·pyr, THF, 82% yield; h. Ti(O-i-Pr)₄, TBHP, L-(+)-DET, 4 ÅMS, DCM; i. K₂CO₃, MeOH; j. PPTS, DCM; 64% yield (3 steps); k. PPTS, 1,1-dimethoxycyclopentane, DCM, 97% yield; l. K₂OsO₄·2H₂O, DHQ-MEQ, K₂CO₃, K₃Fe(CN)₆, MeSO₂NH₂, t-BuOH/H₂O,; 96% yield; dr 10/1; m. PPTS, 1,1-dimethoxycyclopentane, DCM, 82% yield; n. MsCl, NEt₃, DCM, 96%; o. Raney Ni, H₂, EtOH, 88% yield; p. K₂CO₃, MeOH, 78% yield; q. 95, Et₂O/hexane, 85% yield; r. TBSOTf, 2,6-lutidine, DCM, 91% yield; s. DIBAL, Ni(dppp)Cl₂, THF, then NIS; 76% yield; t. DDQ, pH 7 buffer, DCM, 91% yield; u. TESCl, NEt₃, DCM, 95% yield; v. t-BuLi, Et₂O, 71% yield, C-43 epimer ratio 1.7/1; w. TBSOTF, 2,6-lutidine, DCM, 93% yield; x. TBAF/AcOH, THF, 94% yield; y. (COCl)₂, NEt₃, DMSO; z. Ohira-Bestman reagent, MeOH, Cs₂CO₃, 93% yield (2 steps); aa. LHMDS, MeI, THF, quant.; ab. PdCl₂(P-o-tol₃)₂, Bu₃SnH, THF; ac. I₂, DCM, 73% yield (2 steps).

The third and final fragment of AM3 was prepared in four steps from compound 103 (Scheme 3).¹²⁶ A two-step conversion of iodoalcohol 103 afforded iodosulfone 104 quantitatively. Negishi cross coupling of 104 with organozinc 105 gave vinyl stannane 106 in 81% yield. Finally, a Stille cross coupling of stannane 106 with iodotriene 107 (prepared in two steps from 103) yielded the C-53-C-67 fragment 108 in 75% yield.

Scheme 3. Synthesis of the C-53-C-67 Fragment.

a. PTSH, PPh₃, DIAD, THF, 99% yield; b. (NH₄)₆Mo₇O₂₄·4H₂O, aq. H₂O₂, EtOH/THF, quant. c. 105, Pd(PPh₃)₄, THF, 81% yield; d. 107, Pd(PPh₃)₄, CuCl, DMSO/THF, 75% yield.

The end game of the synthesis is outlined in Scheme 4. The final stages the combination of the three fragments 85, 102, and 108. First, the C-1-C-29 fragment 85 was borylated and coupled with the vinyl iodide of the C-30-C-52 fragment 102 through a rigorously optimized Suzuki cross coupling that afforded the C-1-C-52 fragment 109. After oxidative cleavage of the PMB ether, the alcohol was oxidized to the aldehyde 110. Julia-Kocienski olefination between 110 and the C-53-C-67 fragment 108 gave (E)-alkene 111 that was deprotected to afford AM3 (43).

Scheme 4. AM3 Prepared from Compounds 85 and 102.

a. 85: 9-BBN, THF, then 1M aq. Cs₂CO₃ combined with 102, Pd(PPh₃)₄, DMF, 77% yield; b. DDQ, pH 7 buffer, DCM, 75% yield (2 cycles); c. (COCl)₂, NEt₃, DMSO, DCM, quant.; d. KHMDS, THF/HMPA, 75% yield, E/Z = 10/1; e. HF·pyr, MeOH, (CH₂OH)₂, THF, 58% yield (2 cycles).

4.1.3. Amdigenols

Amdigenols comprise long-chain polyethers produced by the marine dinoflagellate Amphidinium sp.,¹³⁰ which was gathered from Ishigaki Island, Okinawa, Japan.¹³¹ In 2012, Inuzuka found that the dinoflagellate Amphidinium sp., attached to the surface of the red algae Digenea simplex, produced a macromolecule comprising 104 carbon atoms with a molecular weight of 2169 g/mol which was characterized as the polyol amdigenol A (112).¹³⁰ In 2014, Inuzuka isolated amdigenols E (113) and G (114) from the same strain.¹³² In 2020, Matsuda isolated amdigenol D.¹³¹ The relative configurational assignment of amdigenol A was determined by rotating frame Overhauser effect spectroscopy (ROESY) correlations.¹³⁰ Amdigenol A showed cytotoxicity against the 3T3-L1 murine adipocyte, with an IC₅₀ value of 59 μg/mL.¹³⁰ The relative configuration of amdigenol D (115) was assigned by NOESY correlations.¹³¹ ROESY correlations only revealed the relative configuration of the tetrahydropyran rings of amdigenols E and G.¹³² These amdigenols were poor inhibitors of human neuroblastoma cell lines IMR-32 growth, and the inhibition of the 113 and 114 at 100 μM were 13% and 21%, respectively.¹³²

4.1.4. Carteraol E

In search of bioactive metabolites from marine microalgae, Huang et al. (2009) collected the dinoflagellate Amphidinium carterae from Taiwan waters and isolated a polyhydroxy-polyene ichthyotoxic compound, carteraol E (116).¹³³ Carteaol E consists of a 69-carbon linear aliphatic chain possessing three tetrahydropyran rings. The relative configurations of the three tetrahydropyran rings were determined by JBCA and ROESY correlations. Carteraol E exhibited a potent ichthyotoxicity with a median lethal dose (LD₅₀) value of 0.28 μM and an antifungal activity against Aspergillus niger at 15 μg/disk.¹³³

4.1.5. Gibbosols

The gibbosols are long-chained polyols that were isolated from Amphidinium gibbosum collected from the South China Sea in 2020.^134,135 Shan Li et al. employed ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) for the analysis of gibbosols A and B.¹³⁴ The absolute configurations of gibbosols A (117),¹³⁴ B (118)¹³⁴ and C (119)¹³⁵ were determined by a combination of periodate degradation of the 1,2-diol groups, ozonolysis of the carbon–carbon double bonds, JBCA, NOE interactions, modified Mosher’s method, Kishi’s universal NMR database, and density functional theory (DFT)-NMR ¹³C chemical-shift (CS) calculations aided by DP4+ CS and statistical analysis.¹³⁴ Gibbosol A displayed mild activation effects on vascular cell adhesion molecule 1 (VCAM-1) expression (64.2 μM), whereas gibbosol B exhibited mild inhibitory activity on VCAM-1 expression, particularly at a concentration of 100.0 μg/mL (62.7 μM).¹³⁴

4.1.6. Karatungiols

Karatungiols A (120) and B (121), two antimicrobial polyol compounds, were obtained from the cultured symbiotic marine dinoflagellate Amphidinium sp. located at Karatung Island, Indonesia by Washida (2006).¹³⁶ These compounds are characterized by a linear C₆₉ chain with a ketocarbonyl functional group, along with 24 or 25 hydroxy groups and two tetrahydropyran rings.¹³⁶ The relative configuration of the stereogenic carbons of the two tetrahydropyran rings of karatungiol A and B was determined by JBCA and ROESY correlations.¹³⁶ Karatungiol A exhibited antifungal activity against Aspergillus niger at 12 μg/disk and antiprotozoan activity against Trichomonas fetus at 1 μg/mL.¹³⁶

4.1.7. Lingshuiols

Lingshuiols (122),¹³⁷ A (123),¹³⁸ and B (124)¹³⁸ are long-chained linear polyhydroxy compounds that were isolated from the cultured Chinese marine dinoflagellate Amphidinium sp. by Guo et al. (2004). The dinoflagellate was collected from Lingshui Bay, Hainan Province, China.^137,138 The relative configuration of the stereogenic centers of the tetrahydropyran rings of lingshuiol (122) was established by NOESY analysis.¹³⁷ Lingshuiol (122) possessed significant cytotoxic activity against A549 cells and the human leukemia cell line HL-60 with an IC₅₀ value of 0.21 and 0.23 μM, respectively.¹³⁷ 24 h exposure to lingshuiol (122) greatly reduced the viability of the hepatocyte in a dose-dependent manner,¹³⁹ the IC₅₀ value was 0.21 ± 0.02 μM.¹³⁹ In the presence of lingshuiol (122), a rapid mitochondrial swelling was found at an IC₉₀ value of 3.21 ± 0.25 μg/mL.¹³⁹ These results suggest that lingshuiol possesses potent permeability of hepatocyte mitochondria, which may be responsible for its cytotoxicity.

4.1.8. Luteophanols

Doi (1997) isolated the dinoflagellate Amphidinium sp. (strain number, Y-52) from the host invertebrate Pseudaphanostoma luteocoloris from Okinawa, Japan, and found a polyhydroxy compound named luteophanol A (125).¹⁴⁰ Luteophanol A exhibited weak antimicrobial activity against Gram-positive bacteria (MIC values: Staphylococcus aureus, 33 μg/mL; Sarcina lutea, 33 μg/mL; Bacillus subtilis, 66 μg/mL).¹⁴⁰ Subsequently, Kubota et al. (1998 and 2005) discovered luteophanols B (126),¹⁴¹ C (127),¹⁴¹ and D (128),¹⁴² compounds that are similar to luteophanol A, from Amphidinium sp. in Okinawa, Japan. The relative configurations of luteophanols B and C were determined based on ROESY and JBCA data.¹⁴¹ The relative configuration of the stereocenters of the two tetrahydropyran rings of luteophanol D was established by ROESY.¹⁴² Luteophanol D exhibited antibacterial activity against Micrococcus luteus (MIC, 33 μg/mL).¹⁴²

4.1.9. Karlotoxins

Karlodinium veneficum (also known as Gymnodinium galatheanum(143)) is a common naked (nonthecate) dinoflagellate species in both mariculture and natural waters and can also form HABs in natural sea areas. It was first isolated from the Hamoaze, off King William Point, South Yard, Devonport, and reported by Ballantine and Abbott in 1956.¹⁴⁴ Outbreaks of K. veneficum often lead to mass die-offs of marine life¹⁴⁵ and also have an impact on human health.¹⁴⁶ The karlotoxins (KmTx’s) comprise long carbon chain compounds that are soluble in H₂O and EtOH and insoluble in CHCl₃, and are produced by mixotrophic strains of the dinoflagellate K. veneficum.(147,148) Deeds et al. (2002) isolated two toxic compounds, KmTx 1 and KmTx 2 (at that time called Tox A and Tox B), produced by K. veneficum from the bloom samples obtained from Chesapeake Bay.¹⁴⁷ They had hemolytic and cytotoxic activities and it was believed that KMnO₄ and CuSO₄ could be used to treat the bloom.¹⁴⁷ Since the isolation of KmTx2 from the eastern US, the global distribution of these dinoflagellates was explored and found to be quite extensive, spanning across North America, Europe, Asia, and Australia. Interestingly, each strain produces slightly different metabolites with varying cytotoxicity and sterol binding profiles.¹⁴⁶ Geographical location, ecological interactions, and other environmental and genetic factors influence the production of these toxins.¹⁴⁹ KmTx 1 (129) was isolated and characterized from K. veneficum found in the Delaware Inland Bays, US.¹⁵⁰ 65-E-chloro-KmTx 1 (130) and 10-O-sulfo-KmTx 1 (131) were isolated from K. veneficum (CCMP 2936) in the Delaware Inland Bays, US.¹⁵¹ KmTx 1, 65-E-chloro-KmTx 1, and 10-O-sulfo-KmTx 1 exhibit hemolytic activity with EC₅₀ values of 63,¹⁵⁰ 56,¹⁵¹ 300 nM,¹⁵¹ respectively. KmTx 2 (132) was isolated from K. veneficum (CCMP 2064) collected off the coast of Georgia, US.¹⁵² Both 4,5-dihydro-KmTx 2 (133) and 4,5-dihydro-dechloro-KmTx 2 (134) were isolated from K. veneficum (strain GM2) collected from the East China Sea.¹⁵³ The absolute configuration of KmTx 2 was determined using JBCA, the Mosher’s method, and a comparison of its degradation products with synthetic controls.^151,152 The absolute configuration of C-49 was revised by DP4+ data analysis.¹⁵⁴ KmTx 2 killed fish in a dose-dependent manner.¹⁵² Mortalities were observed in both zebrafish and sheepshead minnow juveniles at concentrations ranging from 0.1 to 0.5 μg/mL. These concentrations fall within the range of those for KmTx 2 (0.1 μg/mL to 0.8 μg/mL), which is typically found in water samples during fish kills.¹⁵² KmTx 2 exhibited hemolysis with a 50% hemolytic dose (HD₅₀) value of 1.3 ± 0.3 μM.¹⁵⁴ Both 4,5-dihydro-KmTx 2 and 4,5-dihydro-dechloro-KmTx 2 show strong hemolytic activity against erythrocytes extracted from the caudal vein of gilthead seabream (Sparus aurata), with IC₅₀ values at 997 ± 37 and 943 ± 39 ng/mL, respectively.¹⁵³ KmTx 3 (135), 64-E-chloro-KmTx 3 (136) and 10-O-sulfo-KmTx3 (137) were isolated from K. veneficum (CCMP 2936) found in the Delaware Inland Bays, US.¹⁵¹ KmTx 3, 64-E-chloro-KmTx 3, and 10-O-sulfo-KmTx3 exhibited hemolytic activity with EC₅₀ values of 0.2, 0.11, and 2.4 μM, respectively.¹⁵¹ The acute effects of KmTx 1- 3 have been examined in mice.¹⁵⁵ Following intraperitoneal injection of KmTx analogs, temporary lethargy, and elevated respiratory rates were observed shortly after dosing. However, no fatalities were documented in animals administered with 4,5-dihydro-KmTx 2 at doses up to 500 μg/kg or with KmTx 1 or 3 at doses up to 4000 μg/kg.¹⁵⁵ No effects were seen in mice dosed orally with KmTx 1 or 3 at a dose of 4000 μg/kg. The conclusion was that if seafood were to become contaminated with these KmTx’s, the likelihood of causing significant acute intoxication in consumers was considered low.¹⁵⁵ KmTx 8 (138) was isolated from Karlodinium sp. in the US.¹⁵⁴ KmTx 9 (139) was isolated from a closely related species, K.conicum,¹⁵⁴ in the Southern Ocean.¹⁵⁶ The relative configurations of KmTx 8 and 9 were assigned by comparison to KmTx 2. For the portions of KmTx 8 and 9 that were too different for direct comparison by NMR spectroscopy, chemical-shift (CS) calculations were performed to confirm the relative configuration of the new stereogenic centers and link them to the known absolute configuration of C-21 as established for KmTx 2 by degradation chemistry.¹⁵⁴ KmTx 8 exhibited hemolysis with an HD₅₀ value of 0.049 ± 0.004 μM, while compared to KmTx 9 at 3.0 ± 0.3 μM. Leukemia cell lines were sensitive to KmTx 8 SR (50% growth inhibition (GI₅₀) = 0.100 μM) and CCRF-CEM (GI₅₀ = 0.686 μM), non-small cell lung cancer lines HOP-62 (GI₅₀ = 0.986 μM), NCI-H23 (GI₅₀ = 0.903 μM) and HOP-92 (GI₅₀ = 0.501 μM), ovarian cancer cell line IGROV1 (GI₅₀ = 0.631 μM), renal cancer cell line SN12C (GI₅₀ = 1.000 μM), and breast cancer cell line BT-549 (GI₅₀ = 0.501 μM), KmTx 8 was also screened in HeLa cells (GI₅₀ = 369 nM, the tumor growth inhibition value (TGI) = 609 nM, LC₅₀ = 1064 nM).¹⁵⁴ The mode of action of KmTx’s is related to the disruption of the cell membrane by specific binding to cholesterol.¹⁵⁴

Karmitoxin (140) was isolated from K. armiger K-0668 in the Mediterranean Sea.¹⁵⁷ The relative configuration of karmitoxin was assigned by NOESY and JBCA data.¹⁵⁷ Using a calibrated karmitoxin solution, it was found to lyse a rainbow trout gill cell-line (RTgill-W1) in a dose-dependent manner with an LC₅₀ value of 125 ± 1 nM.¹⁵⁷ When evaluating the toxicity of purified karmitoxin in female adult copepods, Acartia tonsa, the 24 h LC₅₀ value was calculated to be 400 ± 100 nM.¹⁵⁷

4.1.10. Ostreols

Ostreopsis spp. are epiphytic, benthic dinoflagellates. Some Ostreopsis species are harmful to marine organisms and also to humans.¹⁵⁸ Ostreol A (141) was isolated from Ostreopsis cf. ovata in the coastal waters of Jeju Island, Korea.^98,159 The relative configuration of ostreol A was determined from ROESY correlations and JBCA data.⁹⁸ Ostreol A exhibited cytotoxicity against Artemia salina with an LD₅₀ value of 0.9 μg/mL.⁹⁸ Ostreol B (142) was isolated from Ostreopsis cf. ovata in the coastal waters of Jeju Island, Korea.¹⁵⁹ The absolute configuration of all stereogenic carbon centers in ostreol B was determined through a combination of the JBCA and ROESY data, and modified Mosher’s method following cleavage of the 1,3-diol functionalities.¹⁵⁹ Ostreol B exhibited moderate cytotoxicity, with IC₅₀ values of 4.8, 0.1, and 0.9 μM against the HepG2, mouse neuroblastoma Neuro-2a cells, and HCT 116 cells, respectively.¹⁵⁹

4.1.11. Symbiopolyol

Symbiopolyol (143) is a long chain compound that was isolated from a symbiotic dinoflagellate Amphidinium sp. (strain KD-056) of the jellyfish Mastigias papua by Hanif (2010) in the Kochi Prefecture, Japan.¹⁶⁰ A partial assignment of the relative configuration of symbiopolyol was determined by ROESY correlations and JBCA data.¹⁶⁰ Symbiopolyol 143 showed VCAM-1 inhibitory activity.¹⁶⁰ The addition of symbiopolyol prior to tumor necrosis factor (TNF)-R stimulation markedly decreased VCAM-1 expression in human umbilical vein endothelial cells (HUVECs) with an IC₅₀ value of 8.23 μg/mL (6.62 μM). Symbiopolyol at 10 μg/mL (8 μM) inhibited TNF-α/interleukin (IL)-4-induced cell adhesion between HUVEC and Ramos cells by 77%.¹⁶⁰

4.1.12. Prorocentrolides

Prorocentrolide A (144) was isolated from Prorocentrum lima at Sesoko Island, Okinawa, Japan.¹⁶¹ The double bond geometry of prorocentrolide A was established by NOESY data.¹⁶¹ Prorocentrolide A acts on both muscle and neuronal nicotinic acetylcholine (ACh) receptors (nAChR), but with a higher affinity for the muscle-type nAChR¹⁶² (affinity constant for muscle-type nAChR = 81.7¹⁶³). Prorocentrolide B (145) was isolated from P. maculosum at Dauphin Island, US.¹⁶⁴ An NOE experiment and the modeling program ConGen were used to establish the relative configuration of some of the cyclic ether moieties, and the hexahydroisoquinoline ring which is present in prorocentrolide B.¹⁶⁴ 4-Hydroxyprorocentrolide (146) and prorocentrolide C (147) were isolated from P. lima (strain no. Plima-YD-5) in Korea.^164,165 The relative configuration of all stereogenic centers of 4-hydroxyprorocentrolide was determined through ROESY correlations and JBCA data.^164,165 Prorocentrolide C exhibits cytotoxicity against HCT 116 and Neuro-2a cells (IC₅₀ = 2.2 and 5.2 μM, respectively).^164,165 The IC₅₀ values of 4-hydroxyprorocentrolide were 17.8 μM for A549 cells and 9.9 μM for HT-29 human colon cancer cells, respectively.¹⁶⁶ The IC₅₀ values of prorocentrolide C were 14.6 μM for A549 cells and 10.5 μM for HT-29 cells, respectively.¹⁶⁶ Spiro-prorocentrimine (148) was isolated from Prorocentrum sp. (PM08) from Taiwan, China.¹⁶⁷ A mouse toxicity assay (intraperitoneal injection [i.p.]) of spiro-prorocentrimine exhibited an LD₉₉ of 2.5 mg/kg.¹⁶⁷

4.1.13. Zooxanthellatoxins

Zooxanthellatoxin-A (ZT-A, 149)¹⁶⁸ was isolated with a congener ZT-B (150)^168,169 from a symbiotic marine dinoflagellate Symbiodinium sp. (strain Y-6) in Okinawa, Japan.¹⁷⁰ A partial assignment of the relative configuration of ZT-A was established through coupling constant (J-values) and NOESY data analysis.¹⁶⁸ A partial assignment of the absolute configuration of ZT-A was done by chemical degradation,¹⁷¹ comparison to synthetic degradation products,^171−174 and Mosher’s method.^171,172 Compound ZT-B contains a 62-membered lactone unit, which was established after comparing its spectroscopic data and degradation products with those of compound ZT-A.¹⁶⁹ The terminal acid portions that comprise the lactone of ZT-B were the same as ZT-A, including the absolute configuration of the stereogenic carbon atoms.¹⁷⁴ ZT-A (2 μM) caused thromboxane A₂ (TXA₂) dependent and genistein-sensitive aggregation in rabbit platelets.¹⁷⁵ ZT-B caused a concentration-dependent contraction of the rabbit isolated aorta at concentrations of 10^–7–10^–5 M.¹⁷⁶ The contraction induced by ZT-B resulted from the entry of calcium ions (Ca²⁺) into the smooth muscle cells, in part, through voltage-operated calcium channels.¹⁷⁷

4.1.14. Aplysiatoxins

Aplysiatoxins (ATXs) were originally isolated from the sea hare Stylocheilus longicauda by Scheuer (1975), which was gathered from Hawaiin waters.⁴⁸ ATX (151) was later isolated from Gracilaria coronopifolia in Japan.¹⁷⁸ The absolute configuration of ATX was determined using optical rotation and chemical degradative data.⁴⁵ Mice died about 2 h after administration IP at 250 μg/kg.¹⁷⁸ Debromoaplysiatoxin (152) was isolated from the cyanobacterium Lyngbya majuscula in the Marshall Islands.¹⁷⁹ Its relative configuration was defined through NOE data, and the absolute configuration via ECD data.¹⁸⁰ ATX and debromoaplysiatoxin were revealed to be tumor promoters.¹⁸¹ The strong tumor promoter ATX triggered the maximal synthesis of early antigen (EA) at concentrations ranging from 5 to 10 ng/mL, while the less potent tumor promoter debromoaplysiatoxin necessitated a concentration of 250 ng/mL to achieve maximal induction.¹⁸¹ Debromoaplysiatoxin exhibited significant anti-Chikungunya virus (CHIKV) activity with an EC₅₀ value of 1.3 μM and selectivity indexes of 10.9.¹⁸² Both 3-methoxyaplysiatoxin (153) and 3-methoxydebromoaplysiatoxin (154) were isolated from Trichodesmium erythraeum in Pulau Seringat Kias, Singapore.¹⁸² Owing to the structural similarities with ATX and debromoaplysiatoxin, the absolute configurations of 3-methoxyaplysiatoxin and 3-methoxydebromoaplysiatoxin were assigned as identical.¹⁸² 3-methoxydebromoaplysiatoxin exhibited significant anti-CHIKV activity with an EC₅₀ value of 2.7 μM and a selectivity index of 9.2.¹⁸² Anhydroaplysiatoxin (155) was isolated from Lyngbya majuscula from Vietnam.¹⁸³ Anhydrodebromoaplysiatoxin (156) was isolated from Gracilaria coronopifolia(184) in Maui, US, and Lyngbya majuscule, Vietnam.¹⁸³ The 1, 10, and 100 μg injections of anhydrodebromoaplysiatoxin caused diarrhea in mice for 1–2, 4–5, and 7–12 h periods, respectively.¹⁸⁴ 2-hydroxyanhydroalysiatoxin (157) was isolated from Moorea producens in Okinawa, Japan.¹⁸⁵ The relative configuration was determined through NOE experiments and JBCA data.¹⁸⁵ Owing to the structural similarities with ATX and anhydroaplysiatoxin, the absolute configuration of 2-hydroxyanhydroalysiatoxin was assumed to be identical but was not verified.¹⁸⁵ Neo-debromoaplysiatoxin A (158) and B (159) were isolated from Lyngbya sp. offshore Hainan Island, China.¹⁸⁶ The absolute configuration of neo-debromoaplysiatoxin A was determined by X-ray diffraction data analysis.¹⁸⁶ The relative configuration of neo-debromoaplysiatoxin B was determined by NOESY data and the absolute configuration was deduced by comparison of the experimental and calculated ECD spectra.¹⁸⁶ Neo-debromoaplysiatoxins A and B were related to structures and potassium channel inhibitory activities and showed selectively against Kv1.5 with IC₅₀ values of 6.94 ± 0.26 and 0.30 ± 0.05 μM, respectively.¹⁸⁶ Neo-debromoaplysiatoxin C (160) was isolated from Lyngbya sp. at the shore of Hainan Island, China.¹⁸⁷ Vicinal J-values and NOESY experiments were used to propose a relative configuration for neo-debromoaplysiatoxin C.¹⁸⁷ The absolute configuration of neo-debromoaplysiatoxin C was determined from the NOESY spectrum and biosynthetic homology.¹⁸⁷ Neo-debromoaplysiatoxin D (161) was isolated from Lyngbya sp. in the South China Sea.¹⁸⁸ The NOESY experiments and vicinal J values were used to establish its relative configuration.¹⁸⁸ It was proposed that neo-debromoaplysiatoxin D and neo-debromoaplysiatoxin A may have the same absolute configuration but data were not provided. Debromoaplysiatoxin D presented significant expression of phosphor-protein kinase C (PKC) δ in HepG2 cells at 10 μM.¹⁸⁸ Neo-debromoaplysiatoxins E (162) and F (163) were isolated from the marine cyanobacterium Lyngbya sp. in the harbor of Sanya, Hainan province, China¹⁸⁹ The relative configurational assignments of neo-debromoaplysiatoxin E and F were determined through NOESY experiments and vicinal J-value analysis.¹⁸⁹ The absolute configurations of neo-debromoaplysiatoxins E and F were determined through analysis of ECD data and gauge-independent atomic orbital (GIAO) NMR shift calculations.¹⁸⁹ Neo-debromoaplysiatoxins E and F exhibited potent blocking activities against Kv1.5 with IC₅₀ values of 1.22 ± 0.22 and 2.85 ± 0.29 μM, respectively.¹⁸⁹ Neo-debromoaplysiatoxins G (164) and H (165) were isolated from Lyngbya sp. from the South China Sea.¹⁹⁰ The relative configurations of neo-debromoaplysiatoxins G and H were assigned through NOESY experiments and JBCA data.¹⁹⁰ Additionally, GIAO NMR CS and DP4+ analyses were used to facilitate assignment of the relative configuration of 164. Neo-debromoaplysiatoxins G and H showed potent blocking action against potassium channel Kv1.5 with IC₅₀ values of 1.79 ± 0.22 and 1.46 ± 0.14 μM, respectively.¹⁹⁰ Neo-debromoaplysiatoxins I (166) and J (167) were isolated from the marine cyanobacterium Lyngbya sp. in the harbor of Sanya, Hainan province, China.¹⁹¹ The relative configurations of neo-debromoaplysiatoxin I and J were determined through NOESY experiments and vicinal J-value analysis, and further assessed by GIAO NMR shift calculations and DP4+ analysis.¹⁹¹ Neo-debromoaplysiatoxins I and J displayed comparable inhibitory activities against the Kv1.5 K⁺ channel with IC₅₀ values of 2.59 ± 0.37 and 1.64 ± 0.15 μM, respectively.¹⁹¹ Neo-debromoaplysiatoxin J exhibited cytotoxicity in the SW480 human colorectal cancer cell line with IC₅₀ values of 4.63 ± 0.20 μM.¹⁹¹ Moreover, neo-debromoaplysiatoxin J showed stronger cytotoxicity against the gastric cancer SGC7901, human colon cancer LoVo, and human lung cancer PC-9 cells, with cell viability less than 20% at 20 μM.¹⁹¹

4.1.15. Manauealide

Manauealides A (168),¹⁹² B (169), and C (170)^184,192 were isolated from Gracilaria coronopifolia from Maui, US. Manauealide B was characterized as a semisynthetic product of debromoaplysiatoxin (152).¹⁹² The relative configurations of manauealide A–C were determined by ROESY and J-value data analysis.¹⁹² The absolute configurations of manauealide A–C were assigned via ECD.¹⁹² A 1 μg injection of either manauealide A or B caused diarrhea in mice for a period of 3–4 h.¹⁹² A 10 μg injection of manauealide A also caused diarrhea in mice for a 3–4 h period, but a 10 μg injection of manauealide B caused diarrhea in mice and death within 12 h.¹⁹² Manauealide C also showed toxicity to mice. Both 1 and 10 μg injections of manauealide C caused diarrhea in mice for 2–3 and 3–4 h periods, respectively.¹⁹²

4.1.16. Aplysiadione and Aplysiaenal

Aplysiadione (171) and aplysiaenal (172) were isolated from Moorea producens in Okinawa, Japan.¹⁹³ Aplysiadione corresponds to a decarboxylated biosynthetic intermediate of the ATXs and aplysiaenal was a shorter chain analog of ATXs.¹⁹³ Aplysiadione was shown to have the same stereostructure as the ATXs except at C-3 (NOE correlations and proton coupling data analysis).¹⁹³ The absolute configuration of aplysiaenal was assigned through total synthesis.¹⁹⁴ The IC₅₀ values of aplysiaenal against HCT 116 cells and L1210 cells were >100 and 93 μM, respectively.¹⁹⁴ Nishikawa and co-workers have completed an asymmetric total synthesis of 172.¹⁹⁴

4.1.17. Nhatrangins

Nhatrangins A (173) and B (174) are truncated derivatives of ATX, isolated alongside ATX, from the cyanobacterium Lyngbya majuscula collected in Vietnam by Orjala in 2010.¹⁸³ The relative configurations of nhatrangins A and B were determined by JBCA and selective NOE data.¹⁸³ The absolute configuration of nhatrangin A was determined through total synthesis.^183,194

4.1.18. Oscillatoxins

Oscillatoxins (OTXs) A (175), 21-bromo-OTX A (176), and 19,21-dibromo OTX A (177) were originally found and isolated from the Oscillatoria nigroviridis and Schizothrix calcicole in Enewetak by Moore in 1978.¹⁹⁵ The absolute configuration of OTX A was determined using specific rotation and NMR spectroscopic data.¹⁹⁶ In contrast, OTX B (OTX B was found to be a 5:1 mixture of two C-4 isomers, namely OTX B1 (178) as major and OTX B2 (179) as the minor component. 31-noroscillatoxin B (180), OTX D (181), and 30-methyloscillatoxin D (182) were isolated from Lyngbya majuscula in Hawaii and Okinawa.¹⁹⁷ The specific rotation, ECD, and NMR spectroscopic data for OTX B1 and its degradation products allowed for the determination of the absolute configuration.¹⁹⁷ The relative configurations of OTX B2 and 31-noroscillatoxin B were determined via NOE data.¹⁹⁷ The relative configurations of OTX D and 30-methyloscillatoxin D was determined by NOE data, and the absolute configurations were determined by ECD.¹⁹⁷ OTX D and 30-methyloscillatoxin D exhibited cytotoxicity against L1210 cells with IC₅₀ values of 48 and 52 μM, respectively.¹⁹⁸ OTX D and 30-methyloscillatoxin D have been the subjects of total synthesis.¹⁹⁹ OTX E (183) and F (184) were isolated from Lyngbya sp. in the South China Sea.¹⁸⁸ The relative configurations of OTX E and F were elucidated by NOESY data and the absolute configurations via ECD data.¹⁸⁸ OTX F exhibited cytotoxicity against L1210 cells with an IC₅₀ value of 29 μM.¹⁹⁸ OTX E exhibited potent blocking activity against Kv1.5 with an IC₅₀ value of 0.79 ± 0.032 μM.¹⁸⁸ The total syntheses of OTX E and F have been reported.¹⁹⁸ 17-Bromooscillatoxin B2 (185), 17-bromo-4-hydroperoxyoscillatoxin B2 (186), 4-hydroperoxyoscillatoxin B2 (187), 17-bromo-4,26-epoxyoscillatoxin B2 (188) and 17-bromo-30-methyloscillatoxin D (189) were isolated from Moorea producens in Okinawa, Japan.¹⁸⁵ The inhibition rates on cytotoxicity (10 μg/mL) of 17-bromooscillatoxin B2, 17-bromo-4-hydroperoxyoscillatoxin B2, 4-hydroperoxyoscillatoxin B2, 17-bromo-4,26-epoxyoscillatoxin B2, and 17-bromo-30-methyloscillatoxin D were 25, 50, 40, 70, and 0%, respectively.¹⁸⁵ The inhibition rates on diatom growth (10 μg/mL) of 17-bromooscillatoxin B2, 17-bromo-4-hydroperoxyoscillatoxin B2, 4-hydroperoxyoscillatoxin B2, 17-bromo-4,26-epoxyoscillatoxin B2, and 17-bromo-30-methyloscillatoxin D were 40, 70, 0, 90, and 30%, respectively.¹⁸⁵ OTX G (190) and H (191) were isolated from M. producens in Okinawa, Japan.^185,200 The inhibition rates on diatom growth (10 μg/mL) of OTX G and H were 35% and 45%, respectively.¹⁸⁵ OTX I (192) was isolated from M. producens (formerly Lyngbya majuscula) in Okinawa, Japan.²⁰¹ OTX I showed moderate toxicity in the cytotoxicity test (IC₅₀ = 4.6 μg/mL) and diatom growth inhibition test (IC₅₀ = 1.2 μg/mL).²⁰⁰ OTX J (193), K (194), L (195), and M (196) were isolated from Lyngbya sp. in the Sanya, Hainan province, China.²⁰² The absolute configuration of OTX J was determined by single-crystal X-ray diffraction data.²⁰² The relative configuration of OTX K-M was determined by NOESY data and was confirmed with GIAO NMR shift calculation and DP4+ analysis.²⁰² OTX J, K, and M exhibited blocking activities against Kv1.5 with IC₅₀ values of 2.61 ± 0.91, 3.86 ± 1.03, and 3.79 ± 1.01 μM, respectively.²⁰²

4.1.18.1. Proposed Biosynthesis of OTX

The biogenesis of this series including OTX I (192) is believed to occur from a single linear polyketide precursor.²⁰⁰

4.1.18.2. Synthesis of OTX D, E, and F and Structurally Related Nhatrangin A

The synthesis of the oscillatoxins has been limited to D (181), E (183), and F (184). The first reported total synthesis was that of OTX-D and its 30-Me (182) variant by Ichihara in 1995.¹⁹⁹ Two decades later, Nishikawa reported the second total synthesis of OTX-D alongside the first total synthesis of OTX-E and F.¹⁹⁸ Nishikawa’s synthesis of OTX-E is shown in Scheme 5. The synthesis began with (−)-β-citronellene (197).²⁰³ Treatment of compound 197 with m-CPBA followed by periodic acid led to aldehyde 198. Addition of 3-methoxyphenylmagnesium bromide to the aldehyde led to a 1:1 diastereomeric mixture of (R) and (S)-alcohols. The diastereomeric mixture was converted to the correct (S)-methyl ether 200 following a Swern oxidation (compound 199), an asymmetric Noyori catalytic hydrogenation, and methylation using iodomethane. Ozonolysis of 200 gave aldehyde 201. Subsequent crotylation with compound 71 to compound 202 and demethylation of the anisole moiety using 2-dimethylaminoethanethiol hydrochloride gave compound 203. Demethylation using 2-dimethylaminoethanethiol hydrochloride was key to reproducibility. Global hydroxy group protection with triethylsilyl triflate (TESOTf) gave compound 204. Ozonolysis of 204 gave aldehyde 205, which was subsequently treated with the lithium acetylide of 206 at −40 °C to provide alcohol 207. Alcohol 207 was oxidized to its corresponding ketone with Dess-Martin periodinane (DMP) and then cyclized under acidic conditions to give deprotected enone 208. Introduction of the methyl acetate moiety was accomplished using potassium 3-methoxy-3-oxopropanoate following Masamune’s protocol²⁰⁴ with successive decarboxylation under basic conditions. Reprotection with triisopropylsilyl chloride (TIPSCl) alongside formation of the (E)-silyl ether gave compound 209. Reduction of the enone moiety with LiBH₄ followed by Lewis acid induced cyclization gave spirocycle 210. Finally, removal of the silyl protecting groups afforded OTX-E (183). OTX-D (181) and 30-Me-OTX-D (182) could be obtained from OTX-E via transesterification with the corresponding alcohol and DMAP in refluxing toluene. Interestingly, OTX-F (184) was also produced as a minor byproduct under the transesterification conditions via an intramolecular β-elimination of the lactone and decarboxylation following the transesterification.

Scheme 5. Nishikawa’s Synthesis of OTX-E.

a. m-CPBA, DCM; b. H₅IO₆, Et₂O; c. 3-methoxyphenylmagnesium bromide, THF, 52% yield (3 steps), dr 1/1; (COCl)₂, NEt₃, DMSO, 92% yield; e. RuCl[(S,S)-Tsden(p-cymene), HCO₂H, NEt₃, 78% yield; f. NaH, MeI, DMF, 98% yield; g. O₃, pyridine, MeOH then PPh₃, 88% yield; h. BF₃-Et₂O, THF then H₂O₂, NaOH; i. HCl-Et₂N(CH₂)₂SH, t-BuOK, DMF, 73% yield (2 steps); j. TESOTf, 2.6-lutidine, DCM, 84% yield; k. O₃, pyridine, MeOH/DCM then PPh₃, 93% yield; l. n-BuLi, – 78 °C THF, then add 206 and warm to −40 °C, 71% yield; m. DMP, DCM, 84% yield; n. Amberlyst-15, DCM, 64% yield; o. CDI, MeCN then CH₂(CO₂Me)(CO₂K), MgCl₂, NEt₃, THF; p. K₂CO₃, MeOH, 72% yield (2 steps); q. TIPSCl, DBU, DCM, 84% yield; r. LiBH₄, Et₂O, 94% yield; s. BF₃-Et₂O, DCM, MS4 Å, 59% yield; t. TBAF, HOAc, THF, 97% yield.

Nhatrangin A (173) is a structurally simplified version of the OTXs. To date, there have been four reports of its total synthesis.^{194,205−207} The most recent report is from Nishikawa in 2023 in which the synthesis of aplysiaenal is also reported.¹⁹⁴ The synthesis of nhatrangin was achieved in five steps from compound 203 (Scheme 6).²⁰³ Following protection of the phenolic group of 203 as a t-butyldimethylsilyl ether (TBSCl, imidazole, DMF, quantative yield), esterification of the secondary hydroxy group of 211 with 212 using Yamaguchi’s conditions gave ester 213. Ozonolysis of 213 to the aldehyde followed by Pinnick oxidation gave carboxylic acid 214. Desilylation afforded nhatrangin A.

Scheme 6. Synthesis of Nhatrangin from Compound 211.

a. TCBCl, NEt₃, DMAP, toluene, 90% yield; b. O₃, DCM/MeOH, – 78 °C then PPh₃, 93% yield; c. NaClO₂, NaH₂PO₄, 2-methyl-2-butene, t-BuOH/H₂O, 92% yield; d. TBAF, THF, 92% yield.

4.1.19. Goniodomins

Goniodomin A (215) was isolated from Alexandrium hiranoi (formerly Goniodoma pseudogoniaulax) collected from a tide pool at Jogashima, Japan, and isolated from A. monilatum from the Gulf Coast of the USA.^208−210 Its relative configuration was determined through analysis of proton J-values and ROESY data.^208,209 The absolute configuration of 215 was assigned using a combination of NMR data comparisons with suitable synthetic model compounds and degradation experiments.²⁰⁹ Goniodomin A inhibited the adenosine triphosphatase (ATPase) activities of atrial myofibrils, myosin B, and reconstituted actomyosin in a concentration-dependent manner.²¹¹ Goniodomin A inhibited atrial myosin B ATPase activity at or greater than a concentration of 10^–9 M. The ATPase activity of actomyosin was enhanced by goniodomin A (10^–8 – 3 × 10^–7 M) but was decreased when the concentration was further increased.²¹¹ Goniodomin A displayed EC₅₀ values of 79.78 and 974.99 nM in a rat liver cell line (Clone 9 cells) and primary cultured rat hepatocytes, respectively.²¹² Goniodomin B (216) was isolated from Alexandrium sp. in Puerto Rico.²¹² The absolute configuration of goniodomin B was revised using NOE data and comparison of NMR data with goniodomin A.²¹³ Goniodomin B was effective in the micromolar range with EC₅₀ values of 1.81 and 7.48 μM for Clone 9 and primary cultured rat hepatocytes, respectively.²¹² Goniodomin C (217) was discovered during the studies of the conversion of goniodomin A to goniodomin B.²¹³ Its absolute configuration was assigned by NOE data and comparison of NMR data with those of goniodomin A.²¹³

4.1.20. Palytoxins, Ostreocins, Ovatoxins

Palytoxin (PlTX) was first investigated in the late 1960’s by three independent research groups studying different environments and species. Attaway examined the zoantharian Palythoa caribaeorum(214) in the Caribbean Sea; Hashimoto focused on Palythoa caribaeorum(215) from Okinawa; and Moore and Scheuer, in 1971, identified palytoxin (218) as the biomolecule responsible for the “limu-make-o-Hana”, a brown moss recognized by local Hawaiians as toxic but later determined to be produced by the zooantharian Palythoa toxica.^216,217 Since then, playtoxin has been reported in various other species in the genus Palythoa with many species commonly found in aquaria as well as in other invertebrates and fishes. The source of this toxin has been attributed to a symbiont dinoflagellate associated with zoantharians, primarily from the genus Ostreopsis.

The structural elucidation of the toxin began with the identification of the largest non peptidic/osidic natural product. In 1981, two independent research groups proposed the planar structure of the molecule from NMR data, one led by Moore²¹⁸ in the USA and the other by Uemura²¹⁹ in Japan. The molecular weight and complexity of this toxic substance (molecular formula C₁₂₉H₂₂₃N₃O₅₄) were unprecedented. The configuration of its 64 stereogenic centers was determined in 1982 by two separate research groups led by Kishi²²⁰ and Moore in the USA.²²¹ The structural and synthetic efforts culminated in the confirmation of the structure by Kishi’s total synthesis of 1989.²²² Recently, the crystal structure of the molecule, when bound to the gate enzyme Na,K-ATPase,²²³ was observed using microED.

PlTX is one of the most potent nonpeptidic toxins found in the marine environment, with an LD₅₀ by intravenous administration of 0.15 μg/kg in mice. It has been implicated in several seafood poisoning incidents involving crabs, fishes, shellfish, etc. Ingestion of the toxin can be fatal to humans, raising serious health concerns.²²⁴ Despite this, there are currently no regulations for PlTX-group toxins in shellfish within the EU or in other regions of the world. However, the European Food Safety Authority (EFSA) has proposed a maximum threshold level of 250 μg/kg shellfish.²²⁵ PlTX is known to cause hemorrhagic effects to mammals and significant impacts to the cardiovascular, kidney, gastrointestinal, and respiratory systems.²²⁶

Several analogues of PlTX have been identified in various species of Ostreopsis. In 1995, the major component of O. siamensis collected in Japan and cultivated was identified by NMR spectroscopy as ostreocin d²²⁷ (OSTD, 219). This C₁₂₇ molecule is a 3,26-didemethyl analogue of PlTX and the configurations of the stereogenic centers remains undetermined. Additional analogues, such as ostreocin a (220) and b (221), have been proposed based on LC-HRMS/MS data.^228,229 Generally, OST analogues are less toxic than PlTX.²³⁰ At the beginning of the 21st century, blooms of a benthic species of Ostreopsis were frequently observed in the Northwestern Mediterranean Sea. This species, identified as Ostreopsis cf. ovata, was extensively studied, and its major metabolite, ovatoxin a (OVTXA, 222) was characterized by NMR data in 2012 by Forino and co-workers.²³¹ Ovatoxins (C₁₂₉) are close analogues of PlTX but exhibit different oxidation patterns. Specifically, OVTX-A lacks hydroxy groups at C-17, C-44, and C-64 while the hydroxyl group of ostreocins at C-42 is present. In the same year, studies using J-coupling constants enabled a complete assignment of the relative configuration of the molecule leading to the revision of the configuration of the C-9 and C-26 methyl groups.²³² While ovatoxins have been linked to human intoxications via aerosols, they are generally less toxic than the PlTX.²³³ Other analogues of ovatoxin a (OVTX-B, C, D, E, G and H and an isobaric PlTX) were characterized as minor components additional hydroxyl or methyl groups.^234−236 OVTX-F, rather than OVTXA, was identified as a major metabolite by LC-MS/MS in another strain of the species Ostreopsis collected from a different location along the Italian coastline.²³⁷ Among six strains of Ostreopsis collected in Cyprus in the Meditrranean Sea, three produced novel ovatoxin analogues named OVTX-I, J1, J2, and K with their structures determined only through LC-HRMS/MS.²³⁸ Additionally, OVTX-L was identified as a new ovatoxin in another strain of O. cf. ovata collected from Italy.²³⁹ Two additional analogues of PlTX named mascarenotoxins A and B were also identified by MS/MS in the extract of O. mascaranensis collected from the Indian Ocean.²⁴⁰ These compounds, with lower molecular weights than PlTX (between 2500 and 2535 Da) exhibit lower hemolytic activities compared to PlTX.

4.1.21. Azaspiracids

In 1995, off the coast of Ireland, a seafood poisoning event involving contaminated mussels caused people to develop DSP-like symptoms including nausea, vomiting, cramping, and diarrhea. This event led to the discovery of azaspiracid (AZA) polyether toxins.²⁴¹ The first structure elucidation report was published three years later in 1998²⁴² and the producing organism, the dinoflagellate Azadinium spinosum, was identified over a decade later in 2009.²⁴³ The azaspiracid structure has been revised four times via total synthesis since its original publication.^244−247 Azaspiracids now constitute a family of over 60 compounds that have been isolated from dinoflagellates and shellfish although many of their putative structures have only been described by MS/MS studies.^248−250 The AZA toxin structures are characterized by having a cyclic amine, a carboxylic acid, and two spiro-ring functionalities. The majority of the differentiation in azaspiracid congeners occurs from substitution at four positions with either -H, -Me, −CO₂H, or −CH₂OH.²⁵¹ Examples shown below include AZA1–5 (223–227), AZA13 (228), AZA17 (229), AZA23 (230), and AZA65 (231). While the underlying mechanism of action has yet to be fully elucidated for AZAs, they have been shown to be potassium blockers²⁵² and calcium level modulators²⁵³ in human cell lines. Animal models have shown teratogenic activity in fish embryos²⁵⁴ and mouse models have shown tumor growth and organ damage.^255−257 Only three of the known AZA derivatives (AZAs 1–3) have current regulation limits corresponding to 160 μg AZA1 equiv kg^–1 of shellfish flesh.²⁵⁸

4.1.22. Stereochemical Analysis of Polyhydroxypolyenes and Polyketides

Assigning the relative and absolute configuration of polyhydroxypolyenes and polyketides is extremely challenging given that many of these structures can have numerous remote stereocenters. Traditionally, single crystal X-ray diffraction has been used for obtaining absolute configuration, however, since these molecules are of a highly flexible nature it makes them challenging candidates for growing a suitable crystal. Other methods have been used in tandem to make these stereochemical assignments, however, obtaining the correct configuration is not always possible. This section will briefly cover the approaches used in the stereochemical analysis of these compounds, namely NMR data analysis, synthetic, biosynthetic approaches, and computational chemistry techniques.

Once the basic two-dimensional skeletal structure has been established using basic structural elucidation techniques such as mass spectrometry and simple 1D and 2D ¹H, and ¹³C NMR experiments, more advanced NMR spectroscopy experiments are performed as needed. These methods are explored first since NMR is a nondestructive analytical method, and the integrity of the compound is preserved. The suite of experiments to establish the 3D structure typically starts with establishing the relative configuration through NOESY or ROESY experiments. These are similar experiments that observe the through-space interactions of protons and are helpful for relative configuration analysis. Following that, the JBCA experiment is used. This method, often referred to as Murata’s Method, was developed in the early 1990s,²⁵⁹ but has been modified significantly over the past 20 years to fit multiple applications especially as it applies to polyketide relative and absolute configurational assignments. This method collects data on both ¹H–¹H and ¹H–¹³C J-values. The magnitude of the coupling constant for each interaction is used to assign stereocenters as either anti or gauche in relation to the remaining carbon chain. This works well for highly substituted chains such as those found in HAB metabolites.^260,261 It is important to note here that no single NMR experiment yields all the required data for JBCA. Rather, it is gathered from multiple experiments depending on the values needed, including but not limited to 1D-¹H, 1D-total correlation spectroscopy (TOCSY), hetero-(ω1)-half-filtered TOCSY (HETLOC), carbon-sorted HETLOC (HSQC-HECADE), J-resolved HMBC, and heteronuclear single quantum multiple bond correlation (HSQMBC). Once all NMR experiments have been exhausted, the next approach is typically chemical degradation methods. These commonly include the modified Mosher’s method to define the absolute configuration of carbinol carbons,¹¹¹ periodate cleavage of 1,2-diol moieties, and/or ozonolysis of carbon–carbon unsaturated bonds. Following the degradation, NMR or GC-MS data analysis of the degradation product is performed. This must be followed up with a stereoselective synthesis of the two or more possible fragments generated.^112,113 The final comparison of the degraded molecule with synthetic standards determines which configuration is correct.¹⁵² While this method yields indisputable proof of absolute configuration, it has many drawbacks such as it is a destructive process, it is challenging to perform degradation reactions and analyze the products on small scale, and in general, it is very time consuming. For these reasons, the method is used less often unless it is the only available option for assigning stereochemistry.

Biosynthetic approaches have continued to provide innovative genomic approaches to the assignment of stereochemistry of complex polyketides, which frequently generate stereogenic centers at double bonds.²⁶² The assignment of a group of macrolactones by the Fenical laboratory illustrates the use of a combination of bioinformatic studies of reductase domains, NMR and X-ray diffraction to characterize the stereochemistry of marinolides.²⁶³

Over the past decade in silico methods have gained popularity as a means to determine the configurations of polyhydroxypolyenes. Advances in the computational power of personal computers and the increased availability of supercomputing clusters have started to meet the increased computing demand required for these complex molecules. The polyhydroxypolyenes and polyketide sections in this article provide numerous examples of computational methods used in conjunction with NMR and synthetic techniques.^{134,154,190,191,202} The most common methods rely on DFT-NMR, ¹H, and ¹³C chemical-shift calculations aided by DP4 or DP4+ statistical analysis. This technique uses GIAO NMR chemical shift calculations. The difference between DP4 and DP4+ is the inclusion of unscaled data and the use of higher levels of theory functionals.²⁶⁴ This is usually done to confirm the relative configuration of new stereogenic centers and correlate them to the known absolute configuration of an adjacent stereocenter. The procedures vary with each type of calculation, but the basic process remains the same. A conformational search is performed on all isomers of the compound or fragments of the compound. Ideally, the entire structure is used for the search, but often due to structure size, flexibility, and computational time-constraints, simplified structures must be carefully chosen. Next, DFT GIAO calculations are completed for all low-energy conformers. The predicted chemical shifts are then combined using Boltzmann weighting function, and finally, DP4 or DP4+ analysis is used to predict the structure with the best fit to the experimental chemical shift values. More detailed computational procedures can be found in previous studies.^{189,265−269} New computational approaches are being developed that will ideally result in faster, more accurate data. These include J-DP4 (combines J-coupling and DP4 analysis) and DP4-AI (attempts to automate the process of structural elucidation, including configuration).^270−272 Computational methods have emerged as a powerful tool to verify the relative and absolute configuration of HAB molecules. As in silico methods improve, this will undoubtedly become the preferred method for assigning relative and absolute configuration for HAB molecules. Indeed, these highly complex HAB toxins have played a critical role in developing and validating new and better tools for the assignment of relative and absolute configuration.

4.2. Alkaloids

4.2.1. Saxitoxins

Saxitoxin (STX, 232) is a neurotoxin isolated from the toxic Alaskan butter clams (Saxidomus giganteus), toxic mussels (Mytilus californianus), and axenic cultures of Gonyaulax catanella found on the coasts of the north Pacific Ocean.²⁷³ The absolute configuration of STX was established by X-ray crystallography.^274,275 The LD₅₀ value of STX in mice was 3.4 μg/kg intravenously, 10 μg/kg intraperitoneally, and 263 μg/kg orally, respectively.²⁷⁶ The enantioselective synthesis of STX has been described.^277−279 Compound 233, 12β-deoxySTX, and 12β-deoxygonyautoxin 5 (234) were isolated from Dolichospermum circinale (TA04) in the Tullaroop reservoir, Victoria, Australia.²⁸⁰ The relative configurations of 12β-deoxySTX and 12β-deoxygonyautoxin 5 were determined by NOE data. The absolute configurations of 12β-deoxySTX and 12β-deoxygonyautoxin 5 were established by comparison with synthetic standards and with NMR spectra of known STXs.²⁸⁰ Compound 235, 11,11-dihydroxysaxitoxin (11,11-dhSTX), was isolated from Alexandrium tamarense off the eastern Canadian coasts.²⁸¹ The absolute configuration of 11,11-dhSTX was established by total synthesis.²⁸² The IC₅₀ value of 11,11-dhSTX had been measured against rat Na_V1.4, and was found to be 2.2 μM.²⁸² De novo synthesis of 11,11-dhSTX has been successfully achieved.²⁸² Neosaxitoxin (NEOSTX) (235) was first isolated as the minor toxin found in the Alaskan butter clam, Saxidomus giganteus, and later as a major toxin in the cultured dinoflagellate, Gonyaulax tamarensis, which caused the North Atlantic PSP.²⁸³ NEOSTX (10 nM or 0.2 nmol/day, 28 days) blocks neuronal voltage-dependent Na⁺ channels and could be an innovative drug to block the polycystic ovary (PCO) phenotype, permitting the rats to ovulate and most likely recover from decreased fertilization capacity that occurs after a sympathetic discharge, such as chronic sympathetic stress, and thus, providing support for the use of this drug as a therapeutic agent for polycystic ovary syndrome (PCOS).²⁸⁴ The LD₅₀ value of NEOSTX by IP injection in mice was 8.9 nmol/kg.²⁸⁵ NEOSTX was shown to be a local long-acting pain blocker.²⁸⁶ NEOSTX (1 μM) significantly inhibited the release of nitric oxide (NO), TNF-α, and expression of inducible nitric oxide synthase (iNOS), IL-1β, and TNF-α in lipopolysaccharide (LPS)-activated macrophages of Karlodinium micrum and Karlodinium sp.^287,288 Decarbamoyl neosaxitoxin (dcNEO) (237) was isolated from Aphanizomenon gracile in Lake Iznik, Turkey.²⁸⁹ dcNEO was revealed to be a hydrolysis product of NEOSTX. Compared to STX, the relative acute toxicity of dcNEO was 0.4 (IP, mouse).²⁹⁰ Gonyautoxin (GTX) 1 (238) was isolated from Gonyaulax sp. at Owase Bay, Japan.²⁹¹ GTX 4 (239) was isolated from Alexandrium tamarense in Hiroshima Bay, Japan.²⁹² The LD₅₀ value of GTX 1 and GTX 4 by IP injection in mice were both 14.6 nmol/kg.²⁸⁵ GTX 2 (240) and 3 (241) were isolated in Gonyaulax tamarensis from the USA.²⁹³ The IC₅₀ value of GTX 2 and 3 have been measured against rat Na_V1.4, and were found to be 22 and 15 nM, respectively.²⁸² The LD₅₀ of GTX 2 and 3 by IP injection in mice were both 36.7 nmol/kg.²⁸⁵ A chiral synthesis of GTX 2²⁸² and GTX 3^282,294 has been reported. Compound (242), 12β-deoxyGTX3, was isolated from Anabaena circinalis of the Tullaroop reservoir, Victoria, Australia.²⁹⁵ The absolute configuration of 12β-deoxyGTX3 was established by comparison with synthetic standards.²⁹⁵ GTX 5 (B1) (243) and 6 (B2) (244) were isolated from Pyrodinium bahamense var. compressa of the Senzaki Prefecture, Japan and Palau island.^296−298 Compared to STX, the relative acute toxicities of B1 and B2 were both 0.1 (IP, mouse).²⁹⁰N-Sulfocarbamoylgonyautoxin-2 (C1) (245) and N-sulfocarbamoylgonyautoxin-3 (C2) (246) were isolated from Protogonyaulax sp. in the Porpoise Islands, Alaska and the northeast Pacific.^299−301 Compared to STX, the relative acute toxicities of C1 and C2 were 0.01 and 0.1 (IP, mouse), respectively.²⁹⁰ Compound (247), 21-sulfo-N-1-hydroxysaxitoxin-11α-hydroxysulfate (C3), and 21-sulfo-N-1-hydroxysaxitoxin-11β-hydroxysulfate (C4) (248) were isolated from cultured Protogonyaulax of the northeast Pacific.²⁶⁰ Compared to STX, the relative acute toxicities of C3 and C4 were 0.01 and 0.1 (IP, mouse), respectively.²⁹⁰ LWTXs 1–6 (247–254) were isolated from Lyngbya wollei collected from the Guntersville Reservoir on the Tennessee River in Alabama.³⁰² The relative configuration of LWTXs 6 was determined by NOE and JBCA data. LWTXs 1–6 caused death in mice at <10, 178, 52, < 10, 346, and <10 MU (Mutagenic Unit per micromole)/μmol, respectively.³⁰² GC1 (255), GC2 (256), and GC3 (257) were isolated from Gymnodinium catenatum in the Derwent Estuary, Tasmania, Australia.³⁰³ The configuration of GC1–3 was established by HPLC analysis using a chiral stationary phase and comparison of NMR data with those of STX, GTX 2, and 3.³⁰³ Preliminary investigations showed that GC1–3 did bind to rat brain sodium channels, demonstrating a biological activity indicative of the known PSP toxins.³⁰³

4.2.2. Gymnodimines

In 1994, when shellfish toxicity was monitored, oysters from the Foveaux Strait, South Island, New Zealand, were found to be toxic at high levels.^304,305 Concurrent blooms of a dinoflagellate, Gymnodinium cf. mikimotoi, were also observed.^306,307 Yasumoto et al. isolated a complex pentacyclic derivative from the dinoflagellate Gymnodinium cf. mikimotoi and named it gymnodimine. Subsequently, more analogs of gymnodimines were identified.^{306,308−310} Gymnodimine (A) (258) was isolated from Gymnodinium cf. mikimotoi in New Zealand.³⁰⁶ The relative configuration of gymnodimine (A) was determined by NOE and JBCA data,³⁰⁶ and the absolute configuration by X-ray crystal structure data analysis.³¹¹ Gymnodimine (A) showed potent ichthyotoxicity against a small freshwater fish, Tanichthys albonubes at 0.1 ppm at pH 8.³⁰⁶ Gymnodimine (A) was relatively toxic by injection with an LD₅₀ value of 96 μg/kg.³¹² Animals either died within 10 min of injection or made a full recovery with no perceptible long-term effects.³¹² Gymnodimine (A) was also toxic after oral administration by gavage (LD₅₀ = 755 μg/kg) but was much less toxic when administered with food.³¹² Gymnodimine (A) in isolated mouse phrenic hemidiaphragm preparations produced concentration- and time-dependent block of twitch responses evoked by nerve stimulation without affecting directly elicited muscle twitches, suggesting that it may block the muscle nAChR.³¹³ Gymnodimine (A) also blocked, in a voltage-independent manner, homomeric human α7 nAChR expressed in Xenopus oocytes.^310,313,314 Alonso et al. (2011) evaluated the effect of long-term exposure of cortical neurons to gymnodimine (A) in the progress of Alzheimer’s disease (AD) pathology.³¹⁵ When cortical neurons were exposed to a concentration of 50 nM of gymnodimine (A), it led to a reduction in the intracellular accumulation of amyloid beta (Aβ) as well as decreased levels of hyperphosphorylated tau protein isoforms, which are recognized by the antibodies AT8 and AT100.³¹⁵ Gymnodimine (A) could potentially serve as a valuable resource in the development of pharmaceuticals aimed at treating neurodegenerative disorders.³¹⁵ The total synthesis of gymnodimine (A) had been reported.³¹⁶ Gymnodimine B (259) was isolated from Gymnodinium sp. in New Zealand.³⁰⁶ The relative configuration of gymnodimine B was established by NOESY and JBCA data.^306,317 Gymnodimine C (260) was isolated from Karenia selliformis in New Zealand³¹⁸ The relative configuration of gymnodimine C was established by NOESY and ROESY data.³¹⁸ Gymnodimine C was found to be a C-18 isomer of gymnodimine B.³¹⁸ Gymnodimine D (261) was isolated from Alexandrium ostenfeldii in the northern Baltic Sea.³¹⁹ The relative configuration of gymnodimine D was established by NOESY data and JBCA.³¹⁹ Compound 262, 16-demethylgymnodimine D and gymnodimine E (263) were isolated from Alexandrium ostenfeldii in Ouwerkerkse Kreek, The Netherlands.³²⁰ The relative configuration of 16-demethylgymnodimine D was determined by NOESY and ROESY experiments, and the absolute configuration via ECD data.³²⁰ Compound 264, 12-methylgymnodimine was isolated from the dinoflagellate Alexandrium peruvianum from the New River in North Carolina.³²¹

4.2.3. Lyngbyatoxins

A highly inflammatory and vesicatory substance, lyngbyatoxin A (teleocidin A-1, 265), was isolated from the lipid extract of a Hawaiian shallow-water variety of M. producens (formerly L. majuscula) Gomont.³²² The absolute configuration of lyngbyatoxin A has been determined by chemical degradation which included ozonolysis.³²³ Lyngbyatoxin A exhibited cytotoxic effects against HeLa, human malignant mesothelioma cell lines ACC-MESO-1 and L1210 cells with IC₅₀ values of 35, 11, and 8.1 μM, respectively.³²⁴ Crustacean lethal activity tests were performed using shrimp (Palaemon paccidents); the LD₁₀₀ value of lyngbyatoxin A was 5 mg/kg.³²⁴ Lyngbyatoxin A was highly toxic to Poecilia vittata (baitfish), killing all fish within 30 min at a concentration in seawater of 0.15 μg/mL.³²² The LD₁₀₀ value of lyngbyatoxin A in mice was about 0.3 mg/kg by intraperitoneal injection.³²² Lyngbyatoxin A (1 μM) contracted the rabbit aorta by an extracellular and intracellular calcium-, endothelium-, and neuron-independent mechanism.³²⁵ Lyngbyatoxin A is a natural tumor promoter and a potent activator of PKC.^326,327 A concentration of 0.1 μM lyngbyatoxin A induced a rapid translocation of PKC from the cytosol to the membrane and subsequently led to a sustained decrease in both cytosolic and membrane PKC activity.³²⁸ The total synthesis of lyngbyatoxin A has been reported.³²⁹ Compound 266, 12-epi-lyngbyatoxin A was isolated from M. producens collected at Kahala Beach, Oahu, HI, US.³²⁴ Compound 267, 2-oxo-3(R)-hydroxylyngbyatoxin A, and 2-oxo-3(R)-hydroxy-13-N-demethyllyngbyatoxin A (268) were isolated from M. producens collected at Kahala Beach, Oahu, HI, US.³³⁰ The relative configurations of compounds 267 and 268 were established by NOESY data and the absolute configurations were determined by specific rotation and ECD data.³³⁰ In cytotoxic assays using L1210 cells, the IC₅₀ values of 2-oxo-3(R)-hydroxylyngbyatoxin A and 2-oxo-3(R)-hydroxy-13-N-demethyllyngbyatoxin A were 98 and 321 μM, respectively.³³⁰ The LD₃₃ values of 2-oxo-3(R)-hydroxylyngbyatoxin A and 2-oxo-3(R)-hydroxy-13-N-demethyllyngbyatoxin A were 89 and 25 mg/kg, respectively.³³⁰ Lyngbyatoxins B (269) and C (270) were isolated from Lyngbya majuscula in Kahala Beach, Oahu, HI, US.³³¹ The absolute configurations of lyngbyatoxin B and C have been determined by ECD data.³³¹ The configuration of the C-25 carbinol carbon for lyngbyatoxin B and the geometry of the Δ²⁴ double bond of lyngbyatoxin C remains to be determined. The 50% inhibition for specific binding of [³H]12-O-tetradecanoylphorbol-13-acetate (³H-TPA) were ED₅₀ = 2.2 μM for lyngbyatoxin B and ED₅₀ = 0.2 μM for lyngbyatoxin C.³³¹

4.2.3.1. Synthesis of Lyngbyatoxin A

The first approach to the total synthesis of lyngbyatoxin A (265) was reported by Natsume in 1987.³³² More recently, Garg reported the synthesis (Scheme 7) through the elaboration of indolactam V (278), an efficient tumor promotor.³²⁹ The synthesis began with conversion of commercially available 5-(benzyloxy)-1H-indole (271) to trimethylsilyl triflate (272) in 7 steps in 62% overall yield. Treatment of compound 272 with CsF generated a benzyne intermediate which was trapped by l-valine derivative 273 to provide compound 274 in good yield. Following a series of transformations to eliminate the primary hydroxy group of compound 274, acrylate 275 was cyclized using ZrCl₄ to provide 265 in high yields. Following C-5 epimerization of 276 to 277, the ester was reduced using LiBH₄ to yield indolactam V (278). Protection of the alcohol as the TBS-ether (279) followed by regioselective bromination gave compound 280 in high yield. Subsequent Negishi cross coupling with zinc enolate 281 provided compound 282 as a 1:1 mixture of diastereomers at the newly formed all-carbon quaternary stereocenter. Reduction of the morpholino amide using Schwartz’s reagent and deprotection of the TBS ether (283) afforded lyngbyatoxin A.

Scheme 7. Synthesis of Lyngbyatoxin A (265).

a. CsF. MeCN, 0 °C to rt, 75% yield; b. H₂, Pd/C, NEt₃, EtOAc; c. Ac₂O, AcOH, rt (87% yield, 2 steps); d. K₂CO₃, DMF, 65 °C, 96% yield; e. ZrCl₄, DCM, 34 °C, 90% yield; f. NaHCO₃, MeOH, 40 °C, 50% yield; g. LiBH₄, THF 0 °C to rt; h. TBSCl, TBAI, imidazole, DMF, rt, 90% yield (2 steps); i. NBS, – 78 °C to −15 °C, THF, 87% yield; j. [P(t-Bu)₃PdBr]₂ (15 mol %), LiBr, THF/PhMe, 80 °C, 75% yield, dr 1/1; k. Cp₂Zr(H)Cl, THF, 50 °C l. BrPPh₃CH₃, t-BuOK, THF, 0 °C to rt, 64% yield (2 steps); m. LiBF₄, CSA, THF, rt, 63% yield.

4.2.4. Spirolides

Spirolides (SPXs) were isolated from Mytilus edulis and Placopecten magellanicus,³³³ and were also found in dinoflagellates.³³⁴ These alkaloid derivatives, produced by Alexandrium ostenfeldii, were shown to be highly toxic metabolites.³³⁴ SPX A (284), C (285), and 13-demethylspirolide C (286) were isolated from A. ostenfeldii in Nova Scotia.³³⁵ The LD₅₀ value of SPX A via intraperitoneal administration in mice was 250 μg/kg.³³⁶ Intraperitoneal injections of 13-demethylspirolide C, using mice and rats, have shown dose-dependent neurotoxicity.³³⁷ The first discovered spirolides showed characteristic symptoms and a highly potent response in the mouse bioassay (LD₁₀₀ = 250 μg/kg via, IP). Death of mice occurred within 7 min after separate IP injections of 5 μg of pure SPX A or SPX C dissolved in 1 mL of 1% Tween 80.³³⁸ Compound 287, 13,19-didemethylspirolide C, and SPX G (288) were isolated from A. ostenfeldii from Limfjorden, Denmark.³³⁷ A minimum lethal dose of 30 μg/kg obtained via intraperitoneal administration in mice was observed for 13,19-didemethylspirolide C.³³⁷ Compound 289, 27-hydroxy-13-demethylspirolide C, and 27-oxo-13,19-didemethylspirolide C (290) were isolated from A. ostenfeldii collected in the North Western Adriatic Sea along the Emilia-Romagna coast, Italy.³³⁹ Its relative configuration was determined by ROESY data.³³⁹ Injections of 0.027 mg/kg of 289 and 0.035 mg/kg of 290 in mice showed some of the typical SPX-induced symptoms, such as hyperextension of the back, arching of the tail, and mild neuro-convulsions alike.³³⁹ SPX H (291) and I (292) were isolated from A. ostenfeldii collected at Ship Harbour, Nova Scotia.³⁴⁰ The relative configuration of SPX H was determined by ROESY data.³⁴⁰ SPX H does not show toxicity in the mouse assay; only transient hunching and lethargy were observed in mice injected intraperitoneally with SPX H at doses up to 2000 μg/kg.³⁴⁰

4.2.5. Pinnatoxins

Pinnatoxins are members of the cyclic imine group of marine toxins.^341,342 In 1995, pinnatoxin A (293) was isolated from the Okinawan bivalve Pinna muricata and its structure was determined.³⁴¹ Pinnatoxins B-D (294–296) isolated from the same source were described in subsequent publications.^343,344 A peridinoid dinoflagellate, named Vulcanodinium rugosum,³⁴⁵ from New Zealand, Australia, and Japan were the first shown to produce pinnatoxins. The presence of pinnatoxins in shellfish has now been described also from Norway and Canada.³⁴⁵ Pinnatoxins are toxic to mammals.^{342,346−348} Pinnatoxin A was isolated from P. muricata from Okinawa, Japan.³⁴¹ Its absolute configuration was deduced based on total synthesis.³⁴⁹ Pinnatoxin A blocked Ach-evoked currents in oocytes expressing the human α7 nAChR with an IC₅₀ value of 0.107 nM³⁵⁰ and showed potent acute toxicity against mice (LD₉₉ = 180 μg/kg).³⁵¹ The total synthesis of pinnatoxin A has been reported.^350,352,353 Pinnatoxins B and C were isolated from P. muricata in Okinawa, Japan.³⁴³ Their relative configurations were determined by NOESY data and the absolute configurations were elucidated through transformation reactions³⁴³ and total synthesis^.354 The LD₉₉ value of a 1:1 mixture of pinnatoxins B and C was 22 μg/kg.³⁵¹ The total syntheses of pinnatoxins B and C have been reported.³⁵⁴ Pinnatoxin D was isolated from P. muricata in Okinawa, Japan.³⁴⁴ Its relative configuration was deduced based on a detailed analysis of NOESY and JBCA data.³⁴⁴ Pinnatoxin D showed the strongest cytotoxicity against P388 cells (IC₅₀ = 2.5 μg/mL).³⁵⁵ Pinnatoxins E (297), F (298), and G (299) were isolated from Crassostrea gigas in South Australia.³⁴² The absolute configurations of pinnatoxins E and F were assumed to be the same as gymnodimine (A).³⁴² The relative configuration of pinnatoxin G was determined by NOESY experiments.³⁴² The LD₅₀ value of pinnatoxin E-G by IP injection in mice were between 16 and 50 μg/kg, respectively.³⁴² Pinnatoxin G also inhibited acetylcholine (Ach)-induced currents in oocytes that expressed the human α7 nicotinic acetylcholine receptor (nAChR) with an IC₅₀ value of 5.06 nM.³⁵⁰ The total synthesis of pinnatoxin G has been reported.³⁵⁰ Pinnatoxin H (300) was isolated from V. rugosum in the South China Sea.³⁴⁶ Its relative configuration was deduced based on a detailed analysis of NOESY and ROESY data³⁴⁶ and the absolute configuration was assumed the same as pinnatoxins E-G, however, no data are provided.³⁴⁶ The LD₅₀ value of pinnatoxin H in mice by intraperitoneal injection was 67 μg/kg and by gavage 163 μg/kg.³⁴⁶ Pinnatoxins bind to the neuronal α7 and muscle-type α1₂βγδ nAChRs.³⁵⁶ The toxins also bind to the nAChR surrogate, acetylcholine-binding protein (AChBP).³⁵⁶ Uniquely, the bulky bridged EF-ketal ring specific to the pinnatoxins extended radially from the interfacial-binding pocket to interact with the sequence-variable loop F and govern nAChR subtype selectivity and central neurotoxicity.³⁵⁶

4.2.5.1. Synthesis of Pinnatoxin A

Pinnatoxin A (293) is a 27-membered carbocyclic polyol containing a cyclic imine unit, 14 stereocenters, a tetrahydrofuran moiety with opposing spirocyclic substituted tetrahydropyrans, and a ketal bridging the EF rings. The first total synthesis of pinnatoxin A was reported by Kishi in 1998.³⁵⁷ The most recent total synthesis was reported by Zakarian in 2011, which improved upon an earlier effort.^350,352 This approach relied on the coupling of the G- and BCD-ring fragments and subsequent macrocyclization. The synthesis of the G-ring fragment 301 began with the esterification of compounds 302 and 303 using Yamaguchi’s protocol to produce compound 304 in 84% yield (Scheme 8). Compounds 302 and 303 were both prepared in 46% overall yield starting from (S)-citronellic acid (nine steps) and d-ribose (10 steps), respectively.³⁵⁸ Formation of the ester enolate of 304 with (S)-lithium amide 305 at low temperatures followed by trapping with trimethylsilyl chloride and subsequent warming led to the Ireland-Claisen rearranged product 307 after an aqueous workup. Reduction of acid 307 with LAH gave alcohol 308 (protected as a benzoyl ester) in good yield over the two steps. Following oxidative removal of the PMB group, ozonolysis of the alkene and reduction with NaBH₄ produced a diol (not shown) which was subjected to a Swern oxidation that produced dialdehyde 309. Acid-catalyzed aldol condensation of dialdehyde 309 established the G-ring in compound 310. The following series of reactions were necessary in order to prepare the G-ring fragment 301 for coupling with the BCD-ring fragment: reduction of the aldehyde, protection of the resulting alcohol as the MOM-ether, cleavage of the TIPS group, Swern oxidation and olefination, deprotection of the benozyl ester and Swern oxidation.

Scheme 8. Synthesis of the G-Ring Fragment 301.

a. NEt₃, DMAP, PhH, 84% yield; b. THF, – 78 °C then TMSCl; c. rt, 12 h then aq. work-up; d. LAH, Et₂O, 81-83% yield (2 steps); e. benzoyl chloride, pyridine; f. DDQ, DCM/H₂O; g. O₃, N-methylmorpholine N-oxide, DCM; h. NaBH₄, EtOH, 77% yield (4 steps); i. (COCl)₂, DMSO, NEt₃, DCM; j. Bn₂NH₂⁺TFA^–, PhMe, 50 °C, 89% yield; k. NaBH₄, EtOH; l. MOMCl, TBAI, i-Pr₂NEt; m. TBAF, THF; n. (COCl)₂, DMSO, NEt₃; o. Ph₃PCH₃Br, KHMDS, 87% yield (5 steps); p. LAH, Et₂O; q. (COCl)₂, DMSO, NEt₃, 87% yield (2 steps).

The BCD-fragment (311) was prepared from advanced intermediate 312, which was prepared in five steps from (E)-4-(methyldiphenylsilyl)pent-3-en-1-ol (Scheme 9). Palladium-catalyzed cross coupling of iodo-312 with the 9-BBN-derivative 313 proceeded in good yield. Dihydroxylation of the olefinic moiety and deprotection and oxidation of the secondary hydroxy groups gave diketone 315. Ketalization with camphor sulfonic acid (CSA) gave bisketal 316, which established the BCD-rings. Protection of both the primary and tertiary hydroxy groups as silyl ethers and reduction of the ester function with DIBAL provided aldehyde 317. Addition of (trimethylsilyl)methyllithium to the formyl group and oxidation to the ketone were followed by a series of reactions to introduce a terminal olefinic function to give compound 318. Finally, compound 318 was converted to the BCD-fragment 311 in two steps.

Scheme 9. Preparation of the BCD-Fragment (311).

a. Pd(dppf)Cl₂, AsPh₃, Cs₂CO₃; b. AD-mix β; c. TBAF; d. (COCl)₂, DMSO; e. CSA, MeOH, then solvent swap to cyclohexane, rt, 48 h, 78% yield; f. TIPSCl, imidazole; g. TESCl, imidazole; h. DIBAL; i. PMBO(CH₂)₃Li; j. DMP, pyridine; k. TMSCH₂Li; l. KHMDS, 76% yield (8 steps); m. DDQ, DCM/H₂O; n. I₂, PPh₃, imidizole, 87% yield (2 steps).

BCD-fragment 311 was coupled with G-fragment 301 in 78% yield after metal–halogen exchange at low temperature (Scheme 10). The TIPS-protected secondary alcohol 319 was converted to macrocyclization precursor 320 following deprotection, oxidation, and Grignard addition. Macrocyclization of 320 to 321 with Grubbs’ catalyst established the macrocyclic core of pinnatoxin A in 75% yield. Introduction of the C-27 methyl group of 322 was accomplished through stereoselective conjugate addition of an enone. The EF ketal rings (compound 323) were formed alongside deprotection of the silyl groups under acidic conditions following introduction of an azido group. The final steps of the synthesis involved formation of the cyclic imine and carboxylic acid that are present in pinnatoxin A.

Scheme 10. Final Stages of the Pinnatoxin A Synthesis.

a. t-BuLi, Et₂O, 1 h, 78% yield; b. TBAF; c. DMP; d. CH₂CHMgBr; e. second-generation Hoveyda-Grubbs (20 mol %); f. DMP, pyridine, DCM; g. MeCu(CN)Li, BF₃-Et₂O, 73% yield (2 steps); h. DDQ, DCM/H₂O; i. Ts₂O, pyridine, DCM; j. NaN₃, DMF, 80 °C, 66% yield (3 steps); k. LiBF₄, IPA/H₂O, 71% yield; l. TEMPO, PhI(OAc)₂; m. NaClO₂, NaH₂PO₄; n.TMSCHN₂; o.H₂, Pd/CaCO₃; p. triethylammonium mesitoate, PhMe, 85 °C, 60 h; q. LiOH THF/H₂O 37-43% (6 steps).

4.2.6. Portimines

Described first in 2013 from the dinoflagellate Vulcanodinium rugosum, the portimines are the smallest member of the cyclic imine family.³⁵⁹ Of the described spirominines, portimine A possesses the weakest in vivo toxicity in animal models, with an IP LD₅₀ value of 1570 μg/kg. However, its activity in in vitro cancer cell models is nearly 100 times more potent than pinnatoxin F with an IC₅₀ value of roughly 3 nM compared to 1 μM. More detailed mechanism of action studies determined it to be one of the most potent naturally produced apoptotic inducers ever discovered.¹³⁶⁰ Portimine B (325) was first described from a laboratory culture of V. rugosum and was shown to be less active than portime A in cancer models.³⁶¹ The total synthesies of portimines A (324) and B in 2023 corrected the structure of portimine B and expanded on the mechanism of action of this class in cancer models including demonstrating efficacy in in vivo tumor clearance models.³⁶² Further detailed structure analysis of portimine B incorporating both the recently described DELTA50 - based DFT NMR chemical shift calculations³⁶³ and the newly published i-HMBC experiment,³⁶⁴ indicated that portimine B adopts a transient hydrate intermediate structure in acidic aqueous conditions commonly used in HPLC and LC-MS analyses which led in part to the structure discrepancies.

4.2.7. Anatoxins

Guanitoxin (GNT), formerly known as anatoxin-a(S) “salivary”, is a naturally occurring cyanotoxin commonly isolated from cyanobacteria, specifically of the genus Anabaena.³⁶⁵ Blooms of Anabaena sp. have been observed in temperate climates of both hemispheres with increasing frequency often causing deaths in livestock, swine, and birds.³⁶⁶Anabaena flos-aquae and Anabaena circinalis produce anatoxin-a (326).^45,366 The toxins are water-soluble alkaloids that have been responsible for a number of incidents of human poisoning, some of which have been fatal.³⁶⁷ Anatoxin-a was isolated from A. flos-aquae NRC44h in Burton Lake, Saskatchewan, Canada.⁴⁵ The structure of Anatoxin-a•HCl was determined by X-ray crystallographic data.³⁶⁸ Anatoxin-a is a potent nicotinic agonist that can produce neuromuscular blockade and death by respiratory arrest.³⁶⁹ It is among the more potent naturally occurring toxins, having an acute IP LD₅₀ value of ca. 375 μg/kg and an oral LD₅₀ value ranging from 1 to 10 mg/kg in mice.³⁶⁹ Anatoxin-a (5 and 20 μg/mL) elicits an increase in peroxidase and glutathione S-transferase (GST) activity in aquatic plants.³⁷⁰ The experiments using ¹³C-labeled precursors revealed that anatoxin-a was biosynthesized from acetate and glutamate.³⁷¹ Homoanatoxin-a (327) and 4-hydroxyhomoanatoxin-a (328) were isolated from Raphidiopsis mediterranea Skuja (strain LBRI 48) in Lake Biwa, Japan.³⁷² The relative and absolute configurations of homoanatoxin-a were assigned by NOE data and by comparison with the NMR spectra of anatoxin-a.³⁷² Homoanatoxin-a was reported to have an LD₅₀ value in mice of 200–250 μg/kg by IP.³⁷² 4-hydroxyhomoanatoxin-a was not toxic to mice up to 2.0 mg/kg by IP.³³⁴ The biosynthesis of homoanatoxin-a parallels anatoxin-a, except for C-12 that was not derived from acetate.³⁷¹ Anatoxin-a(S) (329) was produced by Anabaena flos-aquae clone NRC525–17 and is different from anatoxin-a produced by A. flos-aquae NRC44h.³⁷³ To elucidate the absolute configuration of anatoxin-a(S), R- and S-3 were prepared from d- and l-asparagine, respectively.³⁷³ Purified anatoxin-a(S) had an LD₅₀ (IP, mouse) value of approximately 40–60 μg/kg.³⁷⁴ Anatoxin-a(S) functions as an active site-directed inhibitor of acetylcholinesterase (AChE) at 31 nM. What sets anatoxin-a(S) apart is its ability to form a stable enzyme adduct within the active site of AChE, rendering it resistant to reactivation by oximes due to its unique structural characteristics.³⁷⁵In vivo pretreatment with physostigmine and high concentrations of pralidoxime iodide (2-PAM) were the only effective antagonists against a lethal dose of anatoxin-a(S).³⁷⁵

4.3. Fatty Acid Amides

Prymnesium parvum,³⁷⁶Lyngbya majuscula,³⁷⁷ and Lyngbya semiplena(378) produced a series of unique fatty acid amides. These compounds possessed toxicity to brine shrimp and exhibited modest cannabinoid receptor binding activity.³⁷⁸ Myristamide (330), palmitamide (331), stearamide (332), oleamide (333), erucamide (334), elaidamide (335), and linoleamide (336) were identified from P. parvum recovered from Lake Wichita, TX, US.³⁷⁶ When administered to rats at doses of 10 and 20 mg/kg, oleamide exhibited sleep-inducing properties and caused a dose-dependent reduction in body temperature and locomotor activity.³⁷⁹ Erucamide (5–20 mg/kg) administered orally may alleviate depression- and anxiety-like behaviors in mice.³⁸⁰ Elaidamide was a potent inhibitor of the Xenobiotic-metabolizing enzyme microsomal epoxide hydrolase (mEH) and had a K_i value of 70 nM for recombinant rat mEH.³⁸¹ Linoleamide (336) (EC₅₀ = 20 μM³⁸²) had similar sleep-inducing properties to oleamide, but also affects Ca²⁺ channels and increases intracellular calcium levels in MDCK renal tubular cells by releasing Ca²⁺ stored in the endoplasmic reticulum.³⁷⁶ Semiplenamides A-G (337–343), anandamide-like fatty acid amides, were isolated from a collection of Lyngbya semiplena collected in Papua New Guinea.³⁷⁸ The double bond geometry of semiplenamides A-G was deduced from NOE and JBCA data.³⁷⁸ Their absolute configurations were elucidated by chemical derivatization and analysis using GC-MS data on a chiral stationary phase.³⁷⁸ Semiplenamide A (337) was shown to be a moderate inhibitor (IC₅₀ value, 18.1 ± 3.2 μM) of the anandamide membrane transporter (AMT).³⁷⁸ Semiplenamides A, B, and G are weak cannabinoid receptor 1 (CB₁) agonists. The K_i values of semiplenamides A, B, and G were 19.5 ± 7.8, 18.7 ± 4.6, and 17.9 ± 5.2 μM, respectively.³⁷⁸ In the brine shrimp (Artemia salina) toxicity assay, semiplenamides A-G showed LD₅₀ values of 1.4, 2.5, 1.5, 18, 19, 1.4, and 2.4 μM, respectively.³⁷⁸ Grenadadiene (344), debromogrenadiene (345), and grenadamide (346) were isolated from Lyngbya majuscula collected in Grenada from the Southern Caribbean.³⁷⁷ The relative configurations of grenadadiene, debromogrenadiene, and grenadamide were determined by NOE and JBCA data.³⁷⁷ Grenadamide exhibited modest brine shrimp toxicity (LD₅₀ value, 5 μg/mL) and CB₁ binding activity (K_i value 4.7 μM).³⁷⁷ Compound 347, [(1′Z)-3′-acetoxy-2′-bromo-1′-prop-1′-enyl]-2,5-dimethyldodecanoate, was isolated from Lyngbya sp. collected from Victoria, Australia.³⁸³ The structure of [(1′Z)-3′-acetoxy-2′-bromo-1′-prop-1′-enyl]-2,5-dimethyldodecanoate was established by spectroscopic data analysis and degradation.³⁸³

4.4. Phytoplankton Polyethers

4.4.1. Brevisulcenals

A red tide of Karenia brevisulcata from Wellington Harbour, New Zealand in 1998 was highly toxic to fish and marine invertebrates and also caused respiratory distress in harbor bystanders.³⁸⁴ The ongoing study of the toxins produced by K. brevisulcata identified two suites of complex polyether compounds, including K. brevisulcata toxins and brevisulcatic acids (BSXs).³⁸⁵ An extract of K. brevisulcata showed potent mouse lethality and cytotoxicity, and laboratory cultures of Karenia brevisulcata produced a range of novel lipid-soluble polyether toxins.³⁸⁵ These toxins were named brevisulcenals (KBTs) based on this particular algal species.^386−388 KBTs are high molecular mass polycyclic ethers. Currently, six KBT analogs, namely KBT-A1 (348),³⁸⁶ A2 (349),³⁸⁶ F (350),³⁸⁷ G (351),³⁸⁸ H (352),³⁸⁸ and I (353) have been isolated.³⁸⁸ KBTs possess mammalian toxicity³⁸⁷ and cytotoxicity properties.^385,387 KBT-A1 and A2 were isolated from the red tide dinoflagellate K. brevisulcata (CAWD82) in Wellington Harbour, New Zealand.³⁸⁶ The KBT-A1 and A2 showed cytotoxicity against P388 cells with LC₅₀ values of 10.9 and 3.5 nM, respectively.³⁸⁶ KBT F was isolated from K. brevisulcata (CAWD82) in Wellington Harbour, New Zealand.³⁸⁷ Its relative configuration was determined by combining NOE data and proton J value analysis.³⁸⁷ The mouse lethality of KBT F was estimated to be 0.032 mg/kg.³⁸⁷ KBT F was also toxic against P388 cells at 2.7 nM.³⁸⁷ KBT G, H, and I were isolated from K. brevisulcata (CAWD82) in Wellington Harbour, New Zealand.³⁸⁸ The relative configuration of KBT G was determined by NOE data. Mouse lethality of KBT G was estimated to be 0.032 mg/kg.³⁸⁸ The cytotoxicities of KBT G, H, and I against P388 cells were determined as 0.7, 1.6, and 0.14 nM, respectively.³⁸⁸

4.4.2. Brevisulcatic Acids

A set of water-soluble polycyclic ether toxins was isolated from K. brevisulcata.³⁸⁵ These were named BSXs.³⁸⁵ Currently, the structures of BSX-1 (354),³⁸⁹ 2 (355),³⁹⁰ 4 (356),³⁸⁹ 5 (357),³⁹⁰ and 7 (358)³⁹⁰ have been elucidated.

BSX-1 and BSX-4 were isolated from K. brevisulcata in New Zealand.³⁸⁹ Their relative configurations were defined by NOE correlations and proton J values.³⁸⁹ The mouse IP LD₅₀ values for BSX-1 and BSX-4 were 3.9 and 1.4 mg/kg, respectively.³⁸⁵ In the neuroblastoma cell assay for voltage-gated sodium channel activity (neuro-2a cells in the presence of veratridine and ouabain), BSX-4 displayed a cytotoxicity EC₅₀ value of 20 ng/mL, while that for BSX-1 was 300 ng/mL.³⁸⁹ BSX-4 exhibited hemolysis with an EC₅₀ value of 6660 nM.³⁸⁵ BSX-2, BSX-5, and BSX-7 were isolated from K. brevisulcata in New Zealand.³⁹⁰ The LD₅₀ value (IP, mouse) for lactone BSX-5 was 1.6 mg/kg.³⁸⁵ In the neuroblastoma cell assay for voltage gated sodium channel activity (neuro-2a cells in the presence of veratridine and ouabain), BSX-2 and BSX-5 gave cytotoxicity EC₅₀ values of 370 and 26.9 nM, respectively.³⁸⁵ BSXs are toxic to juvenile salmon and snapper.³⁹¹ Panic, gasping, and loss of balance were observed in all fish exposed to either K. brevis or K. brevisulcata BSXs with effects being apparent soon after exposure to cells.³⁹¹

4.4.3. Brevetoxins

The dinoflagellate Gymnodinium breve is the red tide-causing organism responsible for massive fish kills and human intoxication. The most potent toxin in this class is brevetoxin A (359) or GB-1 toxin and later know as PbTx-1.³⁹² PbTx-1 formed crystals, however, the X-ray data of PbTx-1 would not provide a crystal structure which was successfully resolved by generating the dimethyl acetal. When reported in 1986 it was characterized as the most potent Red-Tide toxin isolated to date with an LC₁₀₀ value of just 4 ng/mL to fish (guppies) and a unique affinity for sodium channels. The brevetoxins (BTXs) are a relatively large family of cyclic polyether metabolites.³⁹³ The initial phytoplankton forms were primarily produced by the dinoflagellate genus Karenia, most notably the species K. brevis (also known as G. breve or Ptychodiscus breve).³⁹³ BTXs were responsible for toxicity in fish, shellfish, marine mammals, birds, and humans.³⁹³ BTX B (PbTx-2,³⁹⁴360) was isolated from P. brevis Davis (G. breve Davis) collected from the Florida coast and the Gulf of Mexico.³⁹⁵ The absolute configuration of BTX B was determined after a five-step synthetic modification (hydrogenation and reduction of the α,β-unsaturated carbonyls, acetylation, dihydroxylation of the C-27-C-28 olefenic unit, and esterification), which introduced two vicinal p-bromobenzoates on the H ring and then applying the nonempirical dibenzoate chirality ECD method and by X-ray diffreaction data.³⁹⁵ The total synthesis of BTX B has been reported.^396−398 BTX B1 (361) was isolated from Austrovenus stutchburyi in New Zealand.³⁹⁹ Its relative configuration was clarified by NOE data and the absolute configuration by ECD spectroscopy.³⁹⁹ The minimum lethal dose of BTX B1 thus isolated was 0.05 mg/kg (IP) in mice.³⁹⁹ BTX B2 (362) was isolated from Perna canaliculus collected in New Zealand.⁴⁰⁰ The relative configuration of BTX B2 was established by NOE data, and the absolute configuration of BTX B2 was assigned by chemical degradation experiments.⁴⁰⁰ The minimum lethal dose of BTX B2 to mice (ddY, male, IP) was estimated to be 306 μg/kg.⁴⁰⁰ BTX B3 (363) was isolated from P. canaliculus in New Zealand.⁴⁰¹ The relative configuration was established by NOE and JBCA data. BTX B3 did not kill mice by IP injection at a dose of 300 μg/kg.⁴⁰¹ BTX B4 (364) was isolated from P. canaliculus in New Zealand.⁴⁰² BTX B4 was shown to be a mixture of N-myristoyl-BTX B2 and N-palmitoyl-BTX B2.⁴⁰² The mouse lethality of BTX B4 by IP injection was estimated to be 0.1 mg/kg.⁴⁰² BTX B5 (365) was isolated from cockle A. stutchburyi in New Zealand.⁴⁰³ The minimum lethal dose was ca. 0.3–0.5 mg/kg (IP) in mice.⁴⁰³ BTX C (366) was isolated from P. brevis from the coast of Florida and in the Gulf of Mexico.⁴⁰⁴ The crude BTXs extract (10 mg/mL, IP, cats⁴⁰⁵) induces central depression of respiratory and cardiac function, spontaneous repeated dose-dependent muscular contractions resulting in fasciculations, twitching or leaping, a precipitous dose-dependent depression in respiratory rate, a bronchoconstrictor response that was central and peripheral in origin, and copious rhinorrhea.⁴⁰⁶ Thermal dysesthesia has also been shown to be a symptom of brevetoxin intoxication in animal models.⁴⁰⁷

4.4.4. Ciguatoxins and Maitotoxins

Ciguatera poisoning (CP) was first documented in the 17th century.⁴⁰⁸ It is a type of foodborne illness caused by consuming fish contaminated with ciguatoxins (CTXs) and/or maitotoxins (MTXs). The term “ciguatera” is derived from cigua, a snail commonly found in the Caribbean Sea. These toxins are produced by microalgae primarily from the genera Gambierdiscus (originally referred to as Goniodoma sp. and Fukuyoa) including species such as Gambierdiscus toxicus. They are predominantly found in the Pacific Ocean, Indian Ocean, Tropical Atlantic Ocean, and more recently, in the Mediterranean and Caribbean seas. Symptoms of ciguatera poisoning can vary widely and typically begin within a few hours to 24 hours after consuming the contaminated fish. These symptoms can include:

• Gastrointestinal symptoms: Nausea, vomiting, diarrhea, and abdominal cramps.

• Neurological symptoms: Numbness or tingling in the mouth, lips, hands, and feet; muscle aches; headache; dizziness; and ataxia (loss of coordination).

• Cardiovascular symptoms: Irregular heartbeats, low blood pressure, and bradycardia (slow heart rate).

• Unusual sensory perceptions: Temperature reversal (cold things feeling hot and vice versa), metallic taste in the mouth, and difficulty distinguishing between hot and cold temperatures.

Although CTXs are highly potent neurotoxins their regulation remains poorly administered worldwide.

CTXs have recently been classified into four main groups: P-CTX1B (367), P-CTX3C (368), and C-CTX1 (369, for Caribbean ciguatoxin⁴⁰⁹) with the structure of the fourth backbone found in species of the Indian Ocean I-CTX not yet elucidated. Despite significant efforts since the 1960s by Scheuer et al.,⁴¹⁰ the planar structure of ciguatoxin (367), the principle toxin of ciguatera in the Pacific Ocean (CTX later named P-CTX1B), was not determined until 1989 by Yasumoto and co-workers⁴¹¹ using NMR spectroscopy on extracts from the moray eel Gymnothorax javanicus. A more lipophilic congener P-CTX4B (370) along with its C-49 epimer P-CTX4A are proposed precursors of ciguatoxin that were identified from G.toxicus, an epibiont of brown seaweeds in the Gambier islands of the tropical Pacific.^412,413 Additional analogues, including P-CTX3C (368) that bears a larger E ring and lacks four carbons on the western side chain, were reported from cultured G. toxicus. The absolute configuration of P-CTX1B (367) was confirmed in 1997⁴¹⁴ by Japanese researchers. Currently, over 18 species of Gambierdiscus are recognized worldwide with at least 11 found to produce CTX metabolites. Cultures of other Pacific species G. australes, G. pacificus and G. polynesiensis (qualified as a CTX-producing super strain) have provided new opportunities to assess the chemical diversity of this group⁴¹⁵ by LC-HRMS/MS and to propose the structures of new analogues. Ciguatoxins have been shown to compete with the brevetoxins (PbTxs) for site-5 on voltage-sensitive sodium channels.^416,417

The structures of ciguatoxins (C-CTX1/2, 369) responsible for food poisoning in the Caribbean Sea were first elucidated by NMR spectroscopy and reported in 1998 by Lewis and co-workers. These structures were identified as C-56 epimers from metabolites isolated from the fish Caranx latus.⁴¹⁸ The elucidation of reduced derivatives at C-56 named C-CTX3/4 (371) was achieved in 2020 using LC-HRMS/MS, also from fish material.⁴¹⁹ A likely precursor of these derivatives, C-CTX5 (372), characterized with a ketocarbonyl at C-3, was identified in 2023 from cultures of two species of Gambierdiscus, G. silvae and G. caribeaus using LC-HRMS/MS.⁴²⁰

Efforts to synthesize this complex family of polyethers culminated in the total synthesis of P-CTX3C in 2001⁴²¹ followed by 51-hydroxy-CTX3C (373).⁴²²

Maitotoxin (MTX, 374) was identified as yet another toxin contributing to Ciguatera Poisoning (CP), with its toxicity measured at 50 ng kg^–1 in mice when administered by IP injection. MTX exhibits a wide range of pharmacological effects with the stimulation of calcium influx into cells playing a central role. The toxin has a molecular weight of 3422 Da (C₁₆₄H₂₅₆O₆₈S₂Na₂), exceeding palytoxin by 748 Da, and it is therefore the largest known biological non-polymer. The planar structure of MTX was initially proposed in 1993 by Yasumoto and co-workers.⁴²³ Subsequent research focused on the structure elucidation of this toxin, including the assignment of the relative configuration of the molecule.⁴²⁴ The structure was revised through extensive efforts aimed at achieving its total synthesis.^425−428

The molecule MTX was found to stimulate Ca²⁺ uptake in cultured NG108–15 neuroblastoma X glioma cells.⁴²⁹ In 1994, three analogues, MTX1, 2 and 3, were isolated from cultures of G. toxicus, but the spectroscopic data were insufficient to determine their structures.⁴³⁰ The structure of MTX3 (375) was finally elucidated in 2019 when the molecule was isolated in sufficient quantity from a culture of the Caribbean species G. belizeanus.⁴³¹ It corresponds to a homologue of the previously described gambierone (376) identified from the same culture.⁴³² Two additional derivatives, MTX2 and MTX4, are present in several species of Gambierdiscus but their structures remain unresolved.⁴³³ Earlier, smaller polyethers were also purified from Gambierdiscus cultures, often as major metabolites though they generally show lower bioactivy compared to CTX or MTX. The planar structures of gambieric acids A–D (377–380) were first characterized in 1993 from cultures of G. toxicus(434) and their absolute confirgurations deduced from chiral spectroscopic techniques in 2000.⁴³⁵ The total synthesis of gambieric acid A in 2012 led to the revision of the absolute configuration.⁴³⁶ These compounds were found to possess potent antifungal activity. In 1993, Yasumoto reported the structure of gambierol (381) from G. toxicus using NMR data⁴³⁷ and its absolute configuration was confirmed in 1999 using Mosher’s analysis.⁴³⁸ The structure was further validated by asymmetric total synthesis in 2002,^439,440 2006,⁴⁴¹ and 2010.⁴⁴² The toxin has been identified as a potent blocker of voltage-gated potassium channels.⁴⁴³

Gambieroxide (382) was isolated and its structure elucidated using NMR data from a culture of G. toxicus from the Pacific in 2013.⁴⁴⁴ Other analogues have been detected in various strains of Gambierdiscus or Fukuyoa using highly sensitive methods such as MS. However, due to the absence of NMR data, their structural features^445,446 remain undetermined.

4.4.5. Prymnesins

Prymnesium parvum is one of the most harmful microalgal red tide species worldwide, posing a serious threat to fish farming especially in brackish waters.^447,448 The organism has been known to produce a potent ichthyotoxin polyether compound named prymnesin.⁴⁴⁹ Based on their ladder-framed polyether backbones, prymnesins can be divided into three main types, i.e., A-, B-, and C-types. The A-, B-, and C-type backbonesare composed of 91, 85, and 83 carbon atoms, respectively.^450,451 Prymnesins have strong hemolytic activity and ichthyotoxicity.^376,452,453 Prymnesin 1 (383) and prymnesin 2 (357) were isolated from P. parvum in Israel.⁴⁵⁴ The configurational assignment of prymnesin 1 was performed by extensively comparing the chemical shifts, J-values, and NOESY data between N-acetylprymnesin-2 (NAPRM2) and N-acetylprymnesin-1 (NAPRM1).^454,455 The relative configuration of prymnesin 2 was assigned by NOEs and JBCA data.⁴⁵⁴ The partial assignment of the absolute configuration was done by comparison between degradation products and synthetic references using fluorimetric HPLC on a chiral stationary phase.^454,456 Prymnesins 1, 2, and their analogs are A-type prymnesins, and both showed extremely potent hemolytic activity.⁴⁵⁴ The hemolytic activity of prymnesin 2 (10.5 nM) was greater than 5000 times that of saponin.³⁷⁶ The LC₅₀ value of prymnesin 2 for the minnow Tanichthys albonubes was 300 nM, but when Ca²⁺ was added the LC₅₀ value was reduced to 3 nM.³⁷⁶ Prymnesin 2 lysed the RTgill-W1 cells with an EC₅₀ value of 0.92 ± 0.05 nM.⁴⁵⁷ Prymnesin-B1 (385) was isolated from P. parvum (K-0081).⁴⁵⁷ The relative configuration of prymnesin-B1 was derived via NOE and JBCA data.⁴⁵⁷ Prymnesin-B1 lysed RTgill-W1 cells with an EC₅₀ value in the nanomolar range (5.98 ± 0.65 nM).⁴⁵⁷ Cytotoxicity in the presence of prymnesins was described as possessing two distinct stages.⁴⁵⁸ The first response to exposure was cell swelling and the second phase was death/lysis.⁴⁵⁹ Prymnesins formed micelles in aqueous solutions, and these aggregates assembled within the plasma membrane of exposed cells to form negatively charged pores that were perm-selective to cations.⁴⁵⁸ Prymnesin extracts possessed neurological toxicity, where it was thought that they interfered with the reuptake of neurotransmitters at the postneuronal synapse.⁴⁵⁸ An intravenous injection of prymnesins stopped heart muscle by rapid depolarization and by altering the membrane permeability to calcium ions.⁴⁵⁸ Meldahl and Fonnum (1993) discovered that the crude extract from P. parvum and P. patelliferum inhibited the sodium-dependent uptake of l-glutamate and γ-aminobutyric acid (GABA) and enhanced the calcium-dependent release of acetylcholine.⁴⁶⁰ The inhibition ratios for the low-affinity vesicular uptake of l-glutamate, GABA, and dopamine were 1/5.8/0.3 and 1/1.7/0.2, respectively.⁴⁶⁰ Mariussen et al. found that prymnesins were responsible for the calcium-dependent release of glutamine at brain synaptosomes.^458,461

4.4.4.1. Biosynthesis of Prymnesins

A cDNA library was constructed from late logphase cultures of the ichthyotoxin-producing haptophyte Prymnesium parvum in 2006.⁴⁶² The majority of tentative unigenes (TUGs), which encompassed 12 out of the 50 most frequently encountered transcripts, were associated with the potential synthesis and secretion of novel proteins. Some of these proteins likely played a role in the production and release of the distinctive prymnesins toxins.⁴⁶² It is relatively certain that prymnesins 1 and 2 are derived from acetate-related metabolism.⁴⁵⁸ Recently, Brad Moore’s lab provided the genomic and trascriptomics evidence for the biosynthetic gene clusters named PKZILLAs for prymnesin-1 (383) from P. parvum. These PKS genes express protein products of 4.7 and 3.2 MDa, respectively and generate 140 and 99 enzyme domains.⁴⁶³ The technical challenges to characterize the very large and complex genomics data, proteins and resulting secondary metabolites followed by their synthesis plays a critical role in expansion of the tools and technologies to characterize and potentially utilize these unique and highly complex products. The report provided the gene sequence for the largest know protein generated by a living organism.

4.4.6. Gymnocins

The dinoflagellate Gymnodinium mikimotoi, collected at Kushimoto Bay, Japan, caused devastating environmental and health ramifications worldwide.⁴⁶⁴ Gymnocins are a series of cytotoxic polyether compounds isolated from the notorious red tide dinoflagellate Karenia (formerly Gymnodinium) mikimotoi.(465) While gymnocins are cytotoxic, they are only weakly toxic to fish.⁴⁶⁵ The reason might arise from the low solubility of the gymnocins in water which prevents them from reaching the fish gills.⁴⁶⁵ Gymnocins-A (386) and B (360) were isolated from G. mikimotoi from Kushimoto Bay, Japan. The relative configuration was assigned by NOE correlations and the ¹H–¹H J-value analysis. The partial assignment of the absolute configuration was elucidated by the modified Mosher’s method.⁴⁶⁵ Gymnocin A displayed cytotoxicity against P388 cells with an IC₅₀ value of 1.3 μg/mL. The structure–activity relationship study revealed that both the α,β-unsaturated formyl unit and the molecular length were required for cytotoxicity.⁴⁶⁶ The total synthesis of gymnocin-A has been reported.^467−469 The relative configuration of gymnocin B was determined by NOE correlations and ¹H–¹H J-value analysis.⁴⁷⁰ The experimentally obtained positive exciton couplet and fluorescence detected circular dichroism (FDCD) of the bis-p-(meso-triphenylporphyrin)-cinnamate (bis-TPPcin) derivative of gymnocin-B were in good agreement with the calculated ECD spectra of the Merck Molecular Force Field (MMFF) optimized structures by employing DeVoe’s coupled oscillator approach, thus establishing the full absolute configuration of gymnocin B.⁴⁷¹ Gymnocin B showed cytotoxicity against P388 cells at 1.7 μg/mL.⁴⁷⁰ The total synthesis of gymnocin B has been reported.⁴⁷²

4.4.5.1. Synthesis of Gymnocin B

Gymnocin B (387) is the second largest contiguous marine ladder polyether compound. It features 15 cyclic ether units (one tetrahydrofuran, nine tetrahydropyran, and five oxepane moieties), a 2-methyl-2-butenal side chain, and one quaternary carbon stereocenter. Jamison reported the first total synthesis of gymnocin B in 2019.⁴⁷² The total synthesis of the related gymnocin A has also been reported.^467−469 Jamison’s strategy for the synthesis of gymnocin B involved a series of separate epoxide ring opening reactions that would prepare the ABCD, FGH, and KLM ring systems (see below for the ring designations) of the natural product. The synthesis of the ABCD ring system (Scheme 11) began from bromoepoxy alkene 388.⁴⁷³ Substition of the bromo with a thiolate group and subsequent oxidation to the sulfone afforded compound 389 in 71% yield. Julia-Kocienski olefination of aldehyde 390 (prepared from 2-deoxyribose)⁴⁷⁴ gave (E)-alkene 391 in 85% yield. Shi epoxidation of the internal olefinic moiety provided the diepoxide 392. Treatment of 392 with phosphate buffer (pH 11) gave compound 393 possessing the CD rings of gymnocin B through an epoxide ring opening cascade in 54% yield. Epoxidation of the terminal olefinic unit and protection of the hydroxy group as a p-methoxybenzyl (PMB) ether gave compound 394. Addition of the mixed cuprate derived from (E)-1-iodo-2-methylpenta-1,4-diene to the oxirane of 394 with subsequent protection of the hydroxy group as a benzyl ether led to compound 395. Shi epoxidation and oxidative cleavage of the PMB ether yielded epoxyalcohol 396. Bromonium ion-initiated epoxide opening led to the formation of the AB rings with the incorrect configuration at C-5. Inversion of C-5 configuration was achieved through a two-step process involving elimination followed by hydroboration/oxidation. Protection of the resulting hydroxy group as a benzyl ether and methanolysis of the ketal group gave compound 398. The ABCD ring system was prepared for coupling following Appel halogenation with elimination to the alkene. Finally, the secondary hydroxy group was protected as a triethylsilyl ether to provide ABCD fragment 399.

Scheme 11. Synthesis of the ABCD Ring System.

a. 1-phenyl-1H-tetrazole-5-thiol, NaH, THF/DMF; b. H₂O₂, (NH₄)₆Mo₇O₂₄·H₂O, NaH₂PO₄ buffer, EtOH, 71% yield (2 steps); c. KHMDS, DMPU, THF, 85% yield, 10:1 E:Z; d. Shi ketone, Bu₄NHSO₄, oxone, K₂CO₃, Na₂B₄O₇, DMM/MeCN/H₂O then TBAF, THF, 65% yield, dr 4/1; e. pH 11 phosphate buffer, 54% yield; f. VO(acac)₂, TBHP, DCM, 68% yield; g. PMPBr, NaH, DMF, 92% yield; h. (E)-1-iodo-2-methylpenta-1,4-diene, n-BuLi, Et₂O, CuCN; i. BnBr, NaH, DMF, 77% yield (2 steps); j. Shi ketone, Bu₄NHSO₄, oxone, K₂CO₃, Na₂B₄O₇, DMM/MeCN/H₂O; k. DDQ, pH 11 phosphate buffer, DCM, 65% yield, dr 6/1; l. NBS, 4 ÅMS, HFIP, 68% yield; m. t-BuOK, THF; n. 9-BBN, THF, NaOH, H₂O₂, 93% yield (2 steps); o. BnBr, NaH, DMF, 99% yield; p. TsOH·H₂O, MeOH/DCM, 89% yield; q. I₂, PPh₃, imidazole, THF, 95% yield; r. TESCl, imidazole, DCM, 97% yield; s. t-BuOK, THF, 95% yield.

The synthesis of the FGH ring system began with known epoxide 400 (from geraniol, Scheme 12).⁴⁷⁵ Ozonolysis to compound 401 followed by Julia-Kocienski olefination with compound 402 gave (E)-alkene 403 in 67% yield over two steps. Shi epoxidation gave triepoxide 404 (76% yield) which was subjected to a BF₃·Et₂O-initiated epoxide ring opening cascade cyclization that formed the GH rings. Subsequent hydroxy protection gave silyl ether 405 in 24% yield over the two steps. Methanolysis of the carbonate followed by oxidation and Wittig olefination gave compound 406. Conjugate reduction using Stryker’s reagent, reduction of the ester, and reoxidation afforded the lactone 407. The FGH fragment 407 was converted to compound 408 through a four-step process requiring ozonolysis, NaBH₄ reduction, silylation, and enol-triflate formation.

Scheme 12. Synthesis of the FGH Ring System.

a. O₃, DCM then PPh₃, 78% yield; b. KHMDS, DMPU, THF, 86% yield, 9:1 E:Z; c. Shi ketone, K₂CO₃, Na₂B₄O₇, DMM/MeCN/H₂O, 76% yield, dr 3/1; d. BF₃·Et₂O, DCM; e. TBSCl, imidazole, DMAP, DMF, 24% yield (2 steps); f. K₂CO₃, MeOH, 88% yield; g. SO₃·pyr, NEt₃, Ph₃PCHCO₂Me, DMSO, DCM, 82% yield; h. [(PPh₃)CuH]₆, THF; i. DIBAL, DCM; j. TEMPO, PIDA, DCM, 79% yield (3 steps); k. O₃, DCM then NaBH₄, MeOH; l. TBDPSCl, imidazole, DCM/DMF, 86% yield (2 steps); m. KHMDS, Comins’ reagent, HMPA, THF, 77% yield.

The synthesis of the KLM ring fragment (Scheme 13) began with alcohol 409.⁴⁷⁶ Swern oxidation, methyl Grignard addition, and reoxidation gave methyl ketone 410. Olefination with compound 411 to compound 412 and subsequent olefination with compound 363 gave triene 413. Shi epoxidation of all three olefinic units gave a triepoxide that was desilylated to compound 414 in 71% yield over the two steps. Treatment of triepoxide 414 with pH 8 phosphate buffer led to an epoxide opening cascade cyclization to generate compound 415 possessing the KLM tetrahydropyran ring system (compound 415) in 23% yield. Following the introduction of PMB-protecting groups, compound 417 was was converted into 418 following methanolysis of the acetonide, the Appel reaction, elimination, and silylation (compound 418).

Scheme 13. Synthesis of the KLM Ring Fragment.

a. (COCl)₂, DMSO, NEt₃, DCM; b. MeMgBr, Et₂O; c. TPAP, NMO, 4 ÅMS, DCM, 42% yield (3 steps); d. KHMDS, DMPU, THF; e. H₂O₂, (NH₄)₆Mo₇O₂₄·H₂O, NaH₂PO₄ buffer, EtOH, 41% yield (2 steps); f. Compound 363, KHMDS, DMPU, THF, 85% yield; g. Shi ketone, Bu₄NHSO₄, Oxone, NaHCO₃, MeCN; h. TBAF, THF, 71% yield (2 steps), dr 2/1; i. pH 8 phosphate buffer, 23% yield; j. DIBAL, PhMe; k. La(OTf)₃, 416, PhMe, 73% yield (2 steps); l. TsOH·H₂O, MeOH, DCM, 94% yield; m. I₂, PPh₃, imidazole, THF, 97% yield; n. TESCl, imidazole, DCM, 99% yield; o. t-BuOK, THF, 97% yield.

The three fragments of gymnocin B were combined through a series of Pd-catalyzed cross couplings with subsequent cyclizations to form the remaining E, N and I rings. The combination of the ABCD- and FGH- to the ABCDEFGH- ring system is illustrated in Scheme 14. Borylation of ABCD fragment 399 with 9-BBN followed by Suzuki cross coupling with FGH fragment 408 gave the ABCDFGH fragment 419. Fragment 419 was subjected to hydroboration of the F ring enolic function, which led to the incorrect configuration of C-20. The hydroxy group was oxidized to a carbonyl and C-20 was epimerized to compound 420. The E ring was formed from compound 420 through a TMSOTf-catalyzed cyclization alongside loss of the two silyl groups in 78% yield. Diol 421 was converted to alkene 422 in an analogous manner as previously described. The preparation of the JKLMNO-ring fragment is also depicted in Scheme 14. Borylation of KLM fragment 418 with 9-BBN was followed by Pd-catalyzed coupling with phosphate 423 that incorporates the O-ring. The O-ring of the KLMO fragment 424 was subjected to DMDO epoxidation, MeMgBr ring opening, and acetylation to afford compound 425 with the correct configuration at C-61. The M-ring compound 425 was subjected to desilylation and oxidation of the secondary hydroxy group to a carbonyl moiety. The N-ring was formed as a hemiacetal 426 after methanolysis of the acetoxy group. Acid-catalyzed removal of the acetal protecting group e of 426 prior to deoxygenation of the N-ring with Et₃SiH to tetraol 427 was necessary. The reductive conditions also cleaved the two PMB ether groups on the K-ring. Reprotection of the 1,3-diol function as an acetonide prior to selective oxidation of the primary hydroxy group (K-ring) to the aldehyde was required. Takai olefination of the formyl group led to alkene 428. The J-ring was established following hydroboration and phenyliodine(III) diacetate (PIDA) oxidation which afforded the lactone 429 in 89% yield over the two steps. The JKLMNO fragment was prepared for coupling as ketene acetal phosphate 430.

Scheme 14. Preparation of the ABCDEFGH Ring System.

a. 399: 9-BBN, THF, Cs₂CO₃, H₂O; then combine with 408, Pd(PPh₃)₄, DMF, 83% yield; b. BH₃, THF, NaOH, H₂O₂, 93% yield, dr >10/1; c. TPAP, NMO, 4 ÅMS, DCM; d. DBU, DCM, 82% yield (2 steps); e. TMSOTf, Et₃SiH, DCM, 78% yield; f. I₂, PPh₃, imidazole, PhH/THF; g. TESCl, imidazole, DMAP, DCM; h. t-BuOK, THF, 89% yield (3 steps); i. 418: 9-BBN, THF, Cs₂CO₃, H₂O; then combine with 423, PdCl₂(dppf)·DCM, DMF, 85% yield; j. DMDO, DCM then MeMgBr; k. Ac₂O, DMAP, pyridine, 77% yield, (2 steps); l. TBAF, THF; m. TPAP, NMO, 4 ÅMS, DCM; n. K₂CO₃, MeOH, 83% yield (3 steps); o. TsOH·H₂O, MeOH/DCM; p. TESOTf, TESH, MeCN; q. TsOH·H₂O, acetone, 65% yield (3 steps); r. DMP, DCM; s. CH₂I₂, CrCl₂, DMF/THF, 76% yield (2 steps); t. 9-BBN, THF, NaOH, H₂O₂; u. TEMPO, PIDA, DCM, 89% yield (2 steps); v. KHMDS, (PhO)₂P(O)Cl, HMPA/THF, 99% yield.

The final stages of the synthesis involved the combination of the individual fragments (Scheme 15). Palladium-catalyzed cross coupling of the ABCDEFGH and JKLMNO fragments 422 and 430 was accomplished by hydroboration, oxidation and epimerization of C-34 in the J ring to afford ketone 431. The I-ring was formed through cyclization of a thioketal followed by reductive desulfurization to afford compound 432, completing the synthesis of the ring systems of gymnocin B. The subsequent steps required the introduction of the 2-methyl-2-butenal side chain. This was achieved following conversion of compound 432 to alkyl iodide 433. Substitution of the iodide with a vinylcuprate, global desilylation with TASF, and olefin metathesis gave gymnocin B.

Scheme 15. Final Stages of the Gymnocin B Synthesis.

a. 422: 9-BBN, THF; Cs₂CO₃, H₂O; then combine with 430. Pd(PPh₃)₄, DMF, 78% yield; b. BH₃, THF, NaOH, H₂O₂; c. TPAP, NMO, 4 ÅMS, DCM; d. DBU, DCM, 72% yield (3 steps); e. EtSH, Zn(OTf)₂, MeNO₂; f. TESOTf, 2,6-lutidine, DCM; g. Ph₃SnH, AIBN, PhMe, 57% yield (3 steps); h. H₂, Pd/C, THF; i. I₂, PPh₃, imidazole, THF; j. TESCl, imidazole, DMAP, DCM/DMF, 65% yield (3 steps); k. vinylmagnesium bromide, CuI, HMPA/THF; l. TASF, THF/DMF; m. methacrolein, Hoveyda-Grubbs II, DCE, 44% yield (3 steps).

4.4.7. Okadaic Acid

Okadaic acid (OA, 434) is a polyether fatty acid first isolated from the marine sponge Halichondria okadai collected in Japan^46,477 and subsequently shown to be produced by several other dinoflagellates such as Prorocentrum sp.^478,479 It concentrates in sponges during filter feeding.⁴⁸⁰ The absolute configuration of OA was determined by X-ray diffraction crystal structure analysis and by comparison to the NMR data of acanthifolicin.⁴⁶ OA was toxic (LC₅₀ = 192 μg/kg; IP mice) and inhibited the growth of KB cells by >30% at 2.5 ng/mL and >80% at 5 ng/mL.⁴⁶ OA induces cell death through the generation of reactive oxygen species (ROS), activation of p38 mitogen-activated protein kinases (MAPKs) and c-Jun N-terminal kinase (JNK), and executed through the mitochondrial-mediated caspase pathway.⁴⁸¹ Additionally, apoptosis induced by OA is a caspase 3-dependent process.⁴⁸² OA is a potent inhibitor of the catalytic subunit of type-2A phosphatase (PP), with IC₅₀ values of 0.5–1 nM.⁴⁸³ With the catalytic subunit of protein PP type-l, IC₅₀ values for OA are between 60 and 500 nM.⁴⁸³ The endogenous PP of smooth muscle myosin B was inhibited by OA with IC₅₀ values of 15–70 nM.⁴⁸³ The partially purified catalytic subunit from myosin B had an IC₅₀ value of 200 nM for OA.⁴⁸³ OA strongly inhibited swelling-stimulated KCl cotransport.⁴⁸⁴ The IC₅₀ for OA was ca. 40 nM, consistent with the involvement of type 1 protein PP in transport activation.⁴⁸⁴ OA exerted a positive inotropic effect in cardiac preparations.⁴⁸⁵ In isolated ³²P-labeled ventricular cardiomyocytes 30 μM OA increased phosphorylation of phospholamban (PLB) and troponin inhibitor (TnI) to 325% and 284% of control, respectively.⁴⁸⁵ The effects of OA could be mediated by increasing the phosphorylation state of several proteins, including PLB, a 23-kDa protein, and TnI.⁴⁸⁵ OA (0.1 μM) induced programmed cell death in the human neuroblastoma cell lines TR14 and human NT2-N neuronal cell line NT2-N.⁴⁸⁶ OA forced differentiated neuronal cells into the mitotic cycle.⁴⁸⁶ The total synthesis of OA has been reported.^487−489 Compound 435, 19-epi-OA, was isolated from the marine dinoflagellate Prorocentrum belizeanum in Spain.⁴⁹⁰ A qualitative analysis of the ROESY experiment established the configuration of all stereogenic carbons.⁴⁹⁰ Compound 435 potently inhibits protein phosphatase 2A (PP2A), showing an IC₅₀ value of 0.47 ± 0.04 nM, virtually equipotent to OA which showed an IC₅₀ of 0.58 ± 0.05 nM.⁴⁹⁰ The inhibitory activity of the toxins versus protein phosphatase 1 (PP1) showed 19-epi-OA presents an IC₅₀ value of 465 ± 52 nM while OA inhibited the enzyme much more potently with an IC₅₀ value of 62 ± 6 nM.⁴⁹⁰ Dinophysistoxin (DTX)-1 (436) was isolated from Prorocentrum lima and Prorocentrum concavum in the Florida Keys, US.⁴⁹¹ The absolute configuration of DTX-1 has been determined by combining the chiral NMR and HPLC reagents with chemical methods.⁴⁹² The LD₅₀ value was 150.4 μg/kg in mice for DTX-1.⁴⁹³ DTX-2 (437) was isolated from Prorocentrum lima, obtained from Northeast Pacific Culture Collection, University of British Columbia, Vancouver, B.C.⁴⁹¹ The relative configuration of DTX-2 was determined by NOESY and ROESY data.⁴⁹⁴ The LD₅₀ for DTX-2 was 338 μg/kg (in mice). The IC₅₀ concentration for DTX-2 inhibition of PP2A was 5.94 ng/mL.⁴⁹⁵ The total synthesis of DTX-2 has been reported.⁴⁹⁶ DTX-3 (438) was isolated from Patinopecten yessoensis in Tsukahama, Ishinomaki Bay, Japan.⁴⁹⁷ DTX-4 (439) was isolated from Prorocentrum lima in Mahone Bay, Nova Scotia, Canada.⁴⁹⁸ The relative configuration of DTX-4 was determined by NOESY.⁴⁹⁸ DTX-4 was toxic to mice with an LD₅₀ = 610 μg/kg, IP.⁴⁹⁸ DTX-5a (440) and −5b (441) were isolated from Prorocentrum maculosum in Mahone Bay, Nova Scotia, Canada.⁴⁹⁹ DTX-5c (442) was isolated from Prorocentrum belizeanum which was obtained from the IEO Vigo collection by courtesy of Santiago Fraga.⁵⁰⁰ The relative configuration of DTX-5c was determined by ROESY and J-value analysis.⁵⁰⁰ DTX-6 (443) was isolated from Prorocentrum lima (strain PLV2).⁵⁰¹ The relative configuration of DTX-6 was the same as OA, according to the ROESY experiment.⁵⁰¹ C₄-diol OA (444),⁵⁰² methyl OA (445),^502,503 norokadanone (446),^502,503 C₆-diol OA (447),⁵⁰² and C₉-diol OA (l) (448)⁵⁰² were isolated from Prorocentrum lima cultured in the laboratory. C₇-diol OA (449),⁴⁹¹ C₈-diol OA (450),^491,502 and C₉-diol OA (k) (451)⁴⁹¹ were isolated from Prorocentrum sp. from the Florida Keys, US. Compound 452, 7-deoxy OA, was isolated from P. lima in New Caledonia.⁵⁰⁴ 7-Deoxy OA inhibited PP2A extracts with an EC₅₀ value of 4.2 × 10^–9 M.⁵⁰⁴ Belizeanic acid (BA, 453) was isolated from Prorocentrun belizeanum.(505) The relative configuration of BA was determined by a combination of JBCA and ROESY data analysis, and comparing the results with the data of OA.⁵⁰⁵ BA was a PP1 inhibitor with an IC₅₀ value of 318 ± 37 nM.⁵⁰⁵

4.4.6.1. Biosynthesis of Okadaic Acid

The origin of the carbon atoms in OA and DTX-1 was investigated by ¹³C-labeled precursor incorporation experiments and the ¹³C-incorporation patterns were analyzed by NMR spectroscopy.^506,507 All carbon atoms except C-37 and C-38 originated from acetate and 16 acetate units were observed to be incorporated into the structures of OA and DTX-1.⁵⁰⁶ Both C-37 and C-38 were derived from glycolate.⁵⁰⁷ It should be noted that all of the pendant methyl groups of OA and DTX-1 were derived from the methyl group of acetate.⁵⁰⁸ Fragments A (C-1 to C-7 and C-44), C (C-11 to C-22 and C-42), and E (C-27 to C-36 and C-39, C-40) were biosynthesized via the classical polyketide pathway.⁵⁰⁸ These incorporation patterns suggested that the cyclization of ether rings C, D, and E occurred via a β-epoxide intermediate at C-22-C-23. The carboxylic acid was formed by the Baeyer–Villiger oxidation.⁵⁰⁹

4.4.8. Prorocentin

Prorocentin (454) was isolated from Prorocentrum lima in Taiwan, China.⁵¹⁰ The relative configuration of prorocentin was determined by NOESY and ¹H–¹H J-value analysis.⁵¹⁰ Prorocentin exhibited inhibitory activity against human colon adenocarcinoma DLD-1 and human malignant melanoma RPMI7951 with IC₅₀ values of 16.7 and 83.6 μg/mL, respectively.⁵¹⁰ The antimicrobial activity against Staphylococcus aureus BRBC 12154 was negative at a dose of 100 μg/mL.⁵¹⁰

4.4.7.1. Synthesis of Prorocentin

Prorocentin (454) is a C₃₉ polyketide. It contains 13 stereogenic centers, two fused hydroxylated tetrahydropyrans with one bearing a spirocyclic dihydropyran moiety, one tetrahydrofuran unit, five olefinic units, an epoxide ring, four hydroxy groups, and five methyls positioned throughout the structure. The original structural assignment of prorocentin was revised following a total synthesis campaign. Katagiri et al. reported the first total synthesis of ent-prorocentin in 2010.⁵¹¹ Fürstner reported the second total synthesis of prorocentin in 2023 and later in the same year reported an optimized second-generation synthesis.^512,513

The synthetic strategy for preparing prorocentin focused on the convergence of three segments of similarly sized carbon count. The synthesis of the C-1-C-9 fragment is shown in Scheme 16. Diyne 455 (prepared from propynylmagnesium bromide and 3-butyn-1-ol) was reduced by LiAlH₄ to enyne 456 in 53% yield. Hydrostannylation of enyne 456 gave vinylstannane 457 in 78% yield. Following O-silylation, a Stille coupling with (E)-(3-iodoallyl)(phenyl)sulfane, catalytic Pd(PPh₃)₄/CuTC and Ph₂PO₂NBu₄ afforded triene 458 as a ca. 10/1 mixture of E/Z isomers. Oxidation of the sulfide to sulfone enabled the separation of the alkene isomers and provided the C-1-C-9 fragment as sulfone 459 in 63% yield over the two steps.

Scheme 16. Synthesis of the C-1-C-9 Fragment.

a. LAH, THF, 53% yield; b. (Bu₃Sn)₂, n-BuLi, CuCN, THF/H₂O, 78% yield; c. TBSCl, imidazole, DMAP, 95% yield; d. (E)-(3-iodoallyl)(phenyl)sulfane, Pd(PPh₃)₄, CuTC, Ph₂PO₂NBu₄, DMF, E/Z = ca. 10/1; e. Na₂WO₄, aq. H₂O₂, MeOH/PhH, 63% yield (2 steps).

The synthesis of the C-10-C-23 fragment is outlined in Scheme 17. Diastereoselective addition to the aldehyde 460 using Carreira’s method failed.⁵¹⁴ Instead, propynylmagnesium bromide was added to aldehyde 460 (prepared in two steps from d-glucose) to form a mixture of diastereomers of propargyl alcohol 461 which was oxidized to the ynone, reduced diastereoselectively, and silylated to form TBS-ether 462. Esterification of 462 with itaconic acid-derived 464 followed by Lindlar reduction of the alkyne gave ester 465. Treatment of ester 465 with Tebbe’s reagent formed the expected enol ether 466 and further underwent intramolecular olefin metathesis to 467 with a 47% yield. It is noteworthy to mention that other olefin metathesis reagents failed to produce 467. Diastereoselective reduction of the dihydropyran 467, desilylation, oxidation of the primary hydroxy group, and subsequent Wittig olefination led to acrylate 468. Reduction of the ester to the allylic alcohol with DIBAL and global silylation gave compound 469. Acetonide 469 was reductively cleaved, and the resulting primary alcohol was oxidized and diastereoselectively propargylated using 470 to form secondary alcohol 471. Acetylation of the alcohol and oxidative cleavage of the PMB-ether led to the C-10-C-23 fragment 472.

Scheme 17. Synthesis of the C-10-C-23 Fragment.

a. CH₃C≡CMgBr, THF, 81% yield, dr 2/1; b. MnO₂, DCM, 61% yield; c. 463 cat., HCO₂H/NEt₃, DCM, 91% yield, dr >20/1; d. TBSCl, imidazole, DCM, 87% yield; e. 464, EDC, DMAP, DCM, 97% yield; f. H₂ (1 atm), Lindlar cat., quinoline, EtOAc, 98% yield; g. Tebbe reagent, THF/PhMe, 47% yield; h. H₂ (1 bar), Pt/C, EtOH, 74-83% yield, dr >20/1; i. TBAF, THF, 81% yield; j. DMP, NaHCO₃, DCM; k. Ph₃P = CHCO₂Me, DCM, 86% yield (2 steps); l. DIBAL, THF, 83% yield; m. TBSOTf, 2,6-lutidine, DCM, 95% yield; n. DIBAL, DCM, 87-93% yield; o. (COCl)₂, NEt₃, DMSO, DCM; p. 470, (R)-3,3′-diBr-BINOL, PhMe, 44-59% yield (2 steps), dr >20/1; q. Ac₂O, pyridine, DMAP, DCM, 90% yield; r. DDQ, pH 7 buffer, DCM, 77% yield.

The synthesis of the C-24-C-36 fragment is outlined in Scheme 18. Alcohol 473 was subjected to an asymmetric Krische allylation reaction to produce homoallylic alcohol 475 in 96% ee. Cobalt-catalyzed Mukaiyama 5-exo-trig oxidative cyclization of 475 led to 2,5-trans-substituted tetrahydrofuran 477 in 72% yield. Primary alcohol 477 was oxidized to the acid, converted to the Weinreb amide, and reacted with 2-methyl-1-propenylmagnesium bromide to form ketone 478. The ketone was subjected to a diastereoselective Luche reduction followed by protection of the secondary alcohol as 2-naphthylmethyl ether. Following this, the alkene was diborylated with B₂pin₂ and catalytic Pt and then oxidized to the corresponding diol 480. Diol 480 was converted to an epoxide and subjected to a ring opening with (1-(trimethylsilyl)vinyl) magnesium bromide to form compound 481. The C-24-C-36 fragment 482 was obtained following iododesilylation and deprotection of the alcohol.

Scheme 18. Synthesis of the C-24-C-36 Fragment.

a. [Ir(cod)Cl]₂, 474, allyl acetate, Cs₂CO₃, 4-Cl-3-NO₂-benzoic acid, THF, 81% yield; b. Co cat. 476, t-BuOOH, iPrOH, O₂, 72% yield, dr >20/1; c. PIDA, TEMPO, MeCN/H₂O, 86% yield; d. MeN(H)OMe-HCl, CDI, DCM, 89% yield; e. 2-methyl-1-propenylmagnesium bromide, THF then DBU cat., DCM, 98% yield; f. NaBH₄, CeCl₃·7H₂O, MeOH, 94% yield, dr >20/1; g. 2-(bromomethyl)naphthalene, NaH, TBAI, THF/DMF, 90% yield; h. Pt(dba)₃ cat., 479 R = mesityl, B₂pin₂, THF, then NaBO₃·4H₂O, THF/H₂O, 76% yield, dr >20/1; i. 1-((2,4,6-triisopropylphenyl)sulfonyl)-1H-imidazole, NaH, THF; j. (1-(trimethylsilyl)vinyl)magnesium bromide, CuCN, THF, 74% yield (2 steps); k. TBSOTf, 2,6-lutidine, DCM, quant.; l. NIS, 2,6-lutidine, HFIP, THF, 80% yield; m. TBAF, HOAc, THF, 83% yield; n. DDQ, pH 7 buffer, DCM, 98% yield.

The end stages of the synthesis (Scheme 19) required combination of the three fragments and preparing the A, C, and D rings. The preparation of the CD rings was addressed first. The C-10-C-23 and C-24-C-36 fragments 472 and 482 were combined through a Sonogashira-Castro coupling reaction to form C-10-C-36 fragment 483 in 91% yield. Alkyne 483 was treated with cationic Au(I) and PPTS to afford BCD ring system 484 in 69% yield. Epoxidation of 484 yielded the epoxide in 84% yield. The protected primary alcohol was desilyated and converted to the primary iodide 485. The C-1-C-9 fragment 459 was alkylated with iodide 485 to form the complete carbon chain of prorocentin. Superhydride reduction of the acetate and sulfone followed by desilylation provided prorocentin.

Scheme 19. End Stages of the Synthesis.

a. Pd₂(dba)₃, CuI, PPh₃, iPr₂NH, 91% yield; b. JohnPhosAu(MeCN)SbF₆ cat., PPTS, DCM, 69% yield; c. TBSOTf, 2,6-lutidine, DCM, 92% yield; d. HF·pyr, pyridine, 92% yield; e. Ti(OiPr)₄, L-(+)-DIPT, cumene hydroperoxide, DCM, 84% yield, dr >20/1; f. I₂, PPh₃, imidazole, DCM, 88% yield; g. 459, n-BuLi, DMPU, THF, 86% yield, dr ca. 3/2; h. LiBHEt₃, THF then [(dppp)PdCl₂], LiBHEt₃, 49% yield; i. HF·pyr, pyridine, THF, 50% yield.

Fürstner’s second-generation synthesis⁵¹³ of prorocentin aimed to improve the synthesis of the central core fragment. The alternative route to the central fragment is outlined in Scheme 20.

Scheme 20. Alternative Route to the Central Fragment.

a. 2-(dimethoxymethyl)naphthalene, TsOH·H₂O, DMF, 86% yield; b. Ag₂CO₃/Celite, PhMe; c. TBSCl, imidazole, DCM, 57% yield (2 steps); d. TBDPSCl, imidazole, DCM; e. DIBAL, pentane; f. Ph₃PCHCO₂Et, PhMe, 59% yield (3 steps); g. DIBAL, THF, 91% yield; h. NapBr, NaH, TBAI, THF, then TBAF, 87% yield; i. I₂, PPh₃, imidazole, THF, 90% yield; j. t-BuLi, THF, 81% yield; k. TBSOTf, Et₃SiH, then PhBCl₂, DCM, MS4 Å, 72% yield; l. (COCl)₂, NEt₃, DMSO, DCM; m. 461, (R)-3,3′-Br-BINOL, PhMe, 68% yield (2 steps); n. Ac₂O, pyridine, DMAP, DCM, 90% yield; o. DDQ, pH 7 buffer, DCM, 67% yield (21% diastereomer).

2-Deoxyglucose (487) was converted to lactone 488 in three steps (acetonide protection, oxidation to the lactone, and silyation). Roche ester 489 was subjected to silylation of the primary hydroxy group, DIBAL reduction to the aldehyde, Wittig olefination with triphenylcarbethoxymethylenephosphorane, reduction to the allylic alcohol with subsequent protection as the 2-methylnaphthyl ether, desilylation, and halogenation to the primary iodide 490. Treating iodide 490 with t-BuLi and combining it with lactone 488 led to compound 491 with an 81% yield. Deoxygenation of the alcohol 491 gave 492. Compound 492 was converted to the new central core fragment 493 in four steps.

4.4.9. Belizeanolide/Belizeanolic Acid

Belizeanolide (494) and belizeanolic acid (495) were isolated from Prorocentrum belizeanum.(515) The partial relative configurational assignment of belizeanolide and belizeanolic acid was determined by NOE, ROESY, and JBCA data.⁵¹⁵ Both belizeanolide and belizeanolic acid showed significant antiproliferative activities.⁵¹⁵ The GI₅₀ (μM) values for belizeanolide were 3.28 ± 0.45 (human epithelial ovarian cancer cell line A2780), 3.23 ± 0.45 (human lung-cancer cells SW1573), 3.23 ± 0.38 (human breast cells HBL100), 3.16 ± 0.40 (T47D breast cancer cells), and 4.58 ± 0.40 (colon adenocarcinoma cell line WiDr).⁵¹⁵ The seco-belizeanolic acid is 10 times more potent than belizeanolide.⁵¹⁵ The GI₅₀ (μM) values for belizeanolic acid were 0.26 ± 0.09 (A2780), 0.31 ± 0.06 (SW1573), 0.32 ± 0.04 (HBL100), 0.40 ± 0.09 (T47D), and 0.41 ± 0.04 (WiDr).⁵¹⁵

4.4.10. Hoffmaniolide

Hoffmaniolide (496), a novel macrolide, was isolated and identified from the marine dinoflagellate Prorocentrum hoffmannianum from Canada.⁵¹⁶ The relative configuration of hoffmaniolide was determined by NOESY and the ¹H–¹H J-value analysis. Hoffmaniolide showed no evidence of cytotoxicity up to 100 μg/mL.⁵¹⁶

4.4.11. Limaol

Limaol (497) was isolated from Prorocentrum lima in Geomundo Island, Korea.⁵¹⁷ The absolute configuration of limaol was completely elucidated based on ROESY correlations, JBCA, and modified Mosher’s ester analysis.⁵¹⁷ Limaol showed cytotoxicity, with IC₅₀ values of 3.7, 7.3, and 9.6 μM against HepG2, HCT 116, and Neuro2a, respectively.⁵¹⁷

4.4.1.1. Synthesis of Limaol

Limaol (497) is a C₄₇ polyol that contains 15 stereocenters, three tetrahydropyran units, one spirocyclic dihydropyran moiety, and an unusual 1,3,5,7-“skipped”-tetraene moiety. Fürstner reported the first total synthesis of limaol in 2021 and later reported a second-generation synthesis in 2024 that greatly improved the material output of the synthesis.^518,519 The strategy for accessing limaol relied on the simplification into three fragments that separated three structural features, namely, the northern skipped tetraene, the central tricycle containing the spirocyclic pyran, and the southern tetrasubstituted pyran. While the skipped tetraene certainly may pose a liability for synthesis due to its thermodynamic instability (unconjugated, 1,1-disubstituted), this motif, however, is rendered metastable as a result of a conformational preference that places the π-bonds nearly orthogonal to one another (i.e., avoidance of syn-pentane-like interaction).⁵¹⁹ The synthesis of the northern fragment is outlined in Scheme 21.

Scheme 21. Synthesis of the C-1-C-11 Fragment.

a. methyl acrylate, DABCO; b. DIBAL, THF, 57% yield (2 steps); c. TBSCl, NaH, THF, 87% yield; d. MsCl, NEt₃, THF, 88% yield; e. LiCl, THF, 98% yield; f. vinylmagnesium bromide, CuI, THF; g. TBDPSCl, imdiazole, DCM, 81% yield (2 steps); h. Hoveyda-Grubbs II, methyl acrylate, DCM, 86% yield; i. TMS-SEt, AlCl₃, THF, 86% yield; j. MeMgBr, CuBr·SMe₂, (S)-(+)-1-[(R)-2-(diphenylphosphino)ferrocenyl]ethyldicyclohexylphosphine, t-BuOMe, 90% yield, dr >20/1; k. TESH, Pd/C, DCM, 85% yield; l. Bestmann-Ohira reagent, K₂CO₃, MeOH, 94% yield; m. 9-I-9-BBN, hexane, then AcOH, quant.; n. Zn, LiCl, THF; o. 500, Pd(PPh₃)₄, THF then TBAF, 76% yield (2 steps); p. Ac₂O, pyridine, DMAP, 96% yield.

Methyl α-bromoacrylate (498) was converted to monoprotected diene 499 in three steps via a Baylis-Hillman reaction followed by reduction and protection. The primary hydroxyl group of 499 was converted to a mesylate that was displaced by LiCl to form primary chloride 500. Separately, vinylmagnesium bromide/Cu(I) was added to propylene oxide 501 and then protected as TBDPS-ether 502 in 81% yield over the two steps. Compound 502 was converted to the β-methyl thioester 503 via olefin metathesis with methyl acrylate, thioesterification, and diastereoselective conjugate addition (dr >20/1). Reductive desulfurization of 503 using triethylsilane provided an aldehyde that was homologated to alkyne 504 using the Bestmann-Ohira reagent in high yield. Alkyne 504 was prepared for coupling to chloride 500 following iodoborylation-deborylation and Zn insertion to give organozinc 505. Combining organozinc 505 with 500 under palladium catalysis formed the corresponding triene. Selective TBS-ether deprotection followed by acetylation gave the C-1-C-11 fragment 506.

The synthesis of the central C-12-C-27 fragment is summarized in Scheme 22. Acetylated glucose 507 was C-glycosylated with allyltrimethylsilane in the presence of borontrifluoride in 56% yield. Following a series of protecting group interconversions, aldehyde 508 was formed following Swern oxidation of the corresponding primary alcohol. BINOL assisted propargylation of aldehyde 508 with allene 461 gave TBS-ether 510 in 96% yield (two steps) after treatment with TBSOTf. Sonogashira-Castro coupling of vinyl iodide 511 with alkyne 510 gave internal alkyne 512 in 93% yield. Cationic gold(I)-catalyzed cyclization and alkene rearrangement of 512 produced spirocycle 513 in 65–78% yield. Finally, osmylation-oxidation provided the C-12-C-27 fragment as aldehyde 514.

Scheme 22. Synthesis of the C-12-C-27 Fragment.

a. Allyltrimethylsilane, BF₃·Et₂O, MeCN, 56% yield; b. NaOMe, MeOH; c. p-methoxybenzaldehyde dimethylacetal, TsOH cat., DMF, 79% yield; d. TBSOTf, 2,6-lutidine, DCM, 86% yield; e. DIBAL, DCM, quant.; f. (COCl)₂, DMSO, NEt₃, DCM, 87% yield; g. 509, (R)-3,3′-diBr-BINOL, PhMe, 96% yield; h. TBSOTf, 2,6-lutidine, DCM, quant.; i. DDQ, DCM/H₂O, 99% yield; j. 511, Pd₂(dba)₃, PPh₃, CuI, iPr₂NH, 93% yield; k. JohnPhos-Au(MeCN)SbF₆., PPTS, DCM, 65-78% yield; o. OsO₄, NaIO₄, 2,6-lutidine, 1,4-dioxane/H₂O, 87-93% yield.

The synthesis of the last C-28-C-40 fragment 520 was accomplished in eight steps from glycal 515 (Scheme 23). Allylation of triacetate 515 with allyltrimethylsilane/TMSOTf proceeded in 57% yield (dr >10/1). The acetoxy groups were cleaved and protected as TBS-ethers (compound 516). Allyl derivative 516 was subjected to olefin metathesis with excess 1-buten-3-ol using a modified Grubbs catalyst. Following silylation, primary alcohol 517 was prepared after selective desilylation using CSA. Excision of the hydroxymethyl group with Pb(OAc)₄ led to acetate 518 in 61% yield. Allylation of 518 with SnCl₄ and trimethylsilylallyl chloride 519 produced the corresponding allyl chloride which after treatment of tributyltinlithium produced the C-28-C-40 fragment as stannane 520.

Scheme 23. Synthesis of the C-28-C-40 Fragment 520.

a. Allyltrimethylsilane, TMSOTf, MeCN, 57% yield, dr >10/1; b. K₂CO₃, MeOH; c. TBSOTf, 2,6-lutidine, DCM, 95% yield (2 steps); d. Modified Grubbs catalyst, 1-buten-3-ol, DCM, 75% yield; e. TBDPSCl, imidazole, DCM, quant.; f. CSA (cat.), MeOH/DCM, 77% yield; g. Pb(OAc)₄, THF, 61% yield; h. SnCl₄, DCM, 83% yield, dr 5/1; i. Bu₃SnLi, THF, 91% yield.

The final stages of the synthesis are shown in Scheme 24. Addition of allylstannane tributylallyltin 520 to C-12-C-27 fragment 514 activated by MgBr₂ led to (R)-C-27 alcohol 522, bearing the incorrect configuration of limaol at C-27. The addition is presumed to proceed through chelate 521 in which the nucleophile adds in a pseudoequatorial fashion. The originally proposed Cram-chelate mode of addition is precluded from occurring due to the inherent steric bias relayed through the bulky TBS-ethers (cf 521). Inversion of the C-27 configuration was accomplished through a Mistunobu reaction sequence followed by silylation to compound 523. Vinylstannane 524 was prepared from the corresponding vinyl triflate of 523 (3/1 mixture of alkene isomers) with (Bu₃Sn)₂CuCNLi₂ at low temperatures. The mixture of stannanes were separated by HPLC. Stille cross coupling between 524 and the C-1-C-11 fragment 506 proceeded in 60% yield to produce silylated-limaol. Desilylation using HF·pyridine proceeded in 32% yield after 11 days to afford limaol.

Scheme 24. Final Stages of the Synthesis.

a. MgBr₂·Et₂O, DCM, 88% yield; b. PPh₃, 4-nitrobenzoic acid, DEAD, PhMe, 67% yield; c. NaOH, MeOH/THF, 91% yield; d. TBSOTf, 2,6-lutidine, DCM, 84% yield; e. Ph₃CK, PhNTf₂, THF; f. (Bu₃Sn)₂CuCNLi₂, THF, 63% yield (3/1 isomers); g. 506, Pd(PPh₃)₄, CuTC, [Bu₄N][Ph₂P(O)O], DMF/THF, 60% yield; h. HF·pyr, THF/pyridine, 32% yield.

Fürstner’s second-generation synthesis of limaol addressed the key issues encountered in the first-generation synthesis, namely, the low yielding desilylation step, direct formation of the correct C-27 configuration, the formation of alkene isomers of stannane 524, and an improved synthesis of the northern fragment. The low yield for the deprotection of silylated-limaol with HF·pyridine is presumed to be a result of the acid-catalyzed ring-opening of the spirocycle over the long reaction time. The steric environment of the caged structure of the molecule (cf. 521) encumbers the approach of the fluoride. Cleavage of the silyl ether at C-20 can only take place upon opening of the spirocycle, a process that leads to the formation of byproducts. Most of the issues inherent in the first-generation approach were addressed through the preparation of a new central fragment precursor 525. Scheme 25 outlines the new approach used in the second-generation route. Cationic gold(I)-catalyzed spirocyclization of 525 proceeded selectivity leading to spirocycle 526 in 79% yield. Desilylation and osmylation-oxidation gave aldehyde 527. Diastereoselective addition of C-28-C-40 fragment 520 to less sterically hindered triacetate 527 afforded alcohol 529 bearing the correct C-27 configuration in 84% yield alongside 12% of the epimer. Direct stannylation of alkyne 529 using (Bu₃Sn)₂CuCNLi in the presence of methanol provided vinylstannane 530. Coupling of stannane 530 with C-1-C-11 fragment 506 followed by ester hydrolysis and desilylation produced limaol. Through this improved route, 277 mg of limaol was produced in a single pass.

Scheme 25. New Approach Used in the Second-Generation Route.

a. JohnPhos-Au(MeCN)SbF₆ (2 mol %), PPTS, DCM, 79% yield; b. AgF, MeCN, 90% yield; c. OsO₄, NaIO₄, dioxane/H₂O, 79% yield; d. 528, 520, DCM, 84% yield (12% C-27-epimer); e. (Bu₃Sn)₂CuCNLi, THF/MeOH, 80% yield; f. 506, Pd₂(dba)₃ cat., LiCl, DMF; g. NaOH, THF/MeOH/H₂O, 70% yield (2 steps); h. TBAF, THF, 99% yield.

4.4.12. Pectenotoxin

Pectenotoxins (PTXs) are a class of lipid-soluble marine toxins with a polyether macrolide structure.⁵²⁰ PTX-1 (531) and PTX-2 (532) were first discovered and identified in 1984 from the digestive glands of farmed scallops Patinopecten yessoensis in Mutsu Bay, Japan.^521,522 It has been shown that many PTXs were formed by metabolism of PTX-2 in shellfish tissues.^523−525 PTX-1 was isolated from Dinophysis fortii in Japan.⁵²² The absolute configuration of PTX-1 was determined by X-ray diffraction crystal analysis.⁵²² Liver cells (cultured for 24 h) that were treated with PTX-1 (0.05–0.5 μg/mL) shrank after 0.5–2 h of exposure to the toxin.⁵²⁶ The marked changes (observed by fluorescent staining) were a result of a reduction of cellular reactivity to antitubulin and phalloidin.⁵²⁶ PTX-2 was isolated from Dinophysis spp. in Honshu, Japan.⁵²² The absolute configuration of PTX-2 was determined by combining chiral NMR or HPLC reagents with chemical methods.⁵²⁷ A clear disrupting effect of PTX-2 (below 200 nM) on the actin cytoskeleton was demonstrated not only by a marked decrease in the levels of cellular F-actin but also by a clear increase in the levels of G-actin in Clone 9 cells.⁵²⁸ The calculated IC₅₀ values of PTX-2 obtained for the rat myoblast cell line (L6) and a human rhabdomyosarcoma cell line (RD) were 60 and 23 ng/mL, respectively,⁵²⁹ PTX-2 was acutely toxic to mice by IP injection (LD₅₀ = 219 μg/kg).⁵³⁰ PTX-2 was lethal to brine shrimps (LC₅₀ < 0.1 μg/mL) during a search for bioactive natural products in sponges.⁵³¹ The total synthesis of PTX-2 has been reported.⁵³² PTX-11 (533) was isolated from Dinophysis acuta in the west coast of South Island, New Zealand.⁵³³ The relative configuration of PTX-11 was determined by NOESY and/or ROESY connectivities and J-value analysis.⁵³³ The absolute configuration of PTX-11 was determined by comparison to NMR data of PTX-2.⁵³³ The LD₅₀ of PTX-11 by mouse intraperitoneal injection was measured as 244 μg/kg.⁵³³ The LD_min of PTX-11 from these experiments was 250 μg/kg.⁵³³ No signs of toxicity were recorded in mice following an oral dose of PTX-11 at 5000 μg/kg.⁵³³ Compound 534, 36S-PTX-12, and 36R-PTX-12 (535) were isolated from Dinophysis spp. in Skjer, Sognefjorden, Norway.⁵³⁴ 36S-PTX-12 and 36R-PTX-12 occurred as a pair of equilibrating diastereoisomers and the relative configurations were determined by NOESY and JBCA data.⁵³⁴ PTX-13 (536) and PTX-14 (537) were isolated from Dinophysis acuta in New Zealand.⁵³⁵

4.4.13. Yessotoxins

Yessotoxins (YTXs) are a class of marine ladder-frame polycyclic ether natural products first isolated in 1986 in Mutsu Bay, Japan, from the digestive glands of the scallops Patinopecten yessoensis after a food intoxication incident.⁵³⁶ Later, Protoceratium reticulatum, Lingulodinium polyedrum, and Gonyaulax spinifera were identified as the true dinoflagellate source that produced these toxins.^536−538 In addition to Japan, YTXs have been identified in shellfish harvested in Europe including Spain, Italy, Norway, the Adriatic Sea, Russia, Chile, and New Zealand.^537,539 YTX (538) was obtained from Protoceratium reticulatum in Japan.⁵³⁸ The absolute configuration of YTX was determined by NMR spectroscopy using a chiral anisotropic reagent, methoxy-(2-naphthyl)acetic acid (2NMA).⁵⁴⁰ YTX inhibited hydrolysis of p-nitrophenyl phosphate by PP2A (IC₅₀ = 0.36 mg/mL).⁵⁴¹ Apoptotic phenotypes in lymphocytes and thymocytes have been reported from mice following lethal (420 μg/kg) or sublethal (10 μg/kg) intraperitoneal injection with YTX.⁵⁴² BC3H1 myoblast cell lines exposed to 100 nM YTX undergo a form of programmed cell death distinct from apoptosis and with features resembling paraptosis.⁵⁴³ YTX exposure at a concentration of 100 nM displayed the characteristics of a ribotoxic stress response in L6 and BC3H1 cells.⁵⁴⁴ An exposure to 100 nM YTX for 12 or 24 h caused an increase of extracellular surface human ether-a-go-go-related gene (hERG) channels.⁵⁴⁵ Furthermore, remarkable bradycardia and hypotension, structural heart alterations, and increased plasma levels of tissue inhibitor of metalloproteinases-1 were observed in rats after four intraperitoneal injections of YTX at doses of 50 or 70 μg/kg that were administered every four days over a period of 15 days.⁵⁴⁵ Additionally, YTX (1 μM) was a phosphodiesterase (PDE) activator in the presence of external Ca²⁺.⁵⁴⁶ At a concentration of 1 nM YTX activates PKC and exhibits favorable effects on the key AD hallmarks, specifically tau and Aβ, in a cellular model derived from fetuses with the triple-transgenic AD (3xTg-AD) condition.⁵⁴⁷ Compound 539, 45,46,47-trinor YTX, was isolated from Protoceratium reticulatum collected at Harima Nada and Yamada Bay in Japan.⁵⁴⁸ Compound 540, 45-hydroxyYTX was isolated from Patinopecten yessoensis in Mutsu Bay, Japan.⁵⁴⁹ The absolute configuration of 45-hydroxyYTX was determined by Mosher’s method.⁵⁵⁰

4.4.12.1. Biosynthesis of YTXs

The biosynthesis of the marine ladder-frame polyether YTX produced by the dinoflagellate Protoceratium reticulatum was investigated by Satake (2011).⁵⁵¹ The ¹³C-labeling experiments showed that the carbon atoms in YTX originated from acetate and glycolate, a methyl group from methionine. The formation of six-membered ring tetrads (rings A-D and H–K) resulted from the repetition of C₃ units (m-m-c), comprising a methyl group from acetate and acetate itself.⁵⁵¹

4.5. Peptides

4.5.1. Microcystin

The cyanobacterium Microcystis aeruginosa has been linked to the death of livestock in both Australia and South Africa due to its production of a family of toxic cyclic peptides in freshwater supplies.⁵⁵² Members of the cyanobacterial genera Microcystis, Oscillatoria, and Anabaena produce cyclic peptides, termed microcystins (MCs), which are potent hepatotoxins.⁵⁵³ These substances were responsible for the deaths of fish, birds, wild animals, and agricultural livestock in many countries where freshwaters contained the toxic cyanobacterial blooms, and adverse effects of the toxins on human health have been recognized.^554,555 MC-LA (also named cyanoginosin-LA,⁵⁵⁶541) was isolated from the cyanobacterium Microcystis aeruginosa in South Australia and South Africa.⁵⁵⁷ The structure of MC-LA was assigned by MS and Edman degradations.⁵⁵⁷ The total synthesis of MC-LA was accomplished.⁵⁵⁸ MC-LR (542 and MC-RR (cyanoviridin RR,^559,560 cyanogenosin-RR,^560,561543) were isolated from the cyanobacterium Microcystis sp. which were collected from Lakes Tsukui and Sagami in Kanagawa prefecture in Japan.^562,563 The relative configurations of MC-LR and MC-RR were established by ROESY analysis.⁵⁶² The absolute configuration of MC-LR was established by Marfey’s method with MS.⁵⁶⁴ The absolute configuration of MC-RR was established by chiral-phase HPLC methods,⁵⁵⁹ chemical degradation,⁵⁶¹ and the comparison of ECD data between synthetic fragments and natural products.⁵⁶⁰ Oral and intraperitoneal administration of the MCs to mammals caused extensive loss of hepatic lobular and sinusoidal architecture with hepatocyte necrosis and hepatic hemorrhage.⁵⁵⁴ A concentration of 1–2 μg of MC-LR constitutes a lethal intraperitoneal dose to mice, with most of the toxin accumulating in the liver and death occurring in about 60 min from hemodynamic shock and heart failure.⁵⁵⁴ At the cellular level, MC-LR causes a rapid, characteristic hepatocyte plasma membrane bleb formation and loss of microvilli with a major reorganization of microfilaments as determined by electron microscopy and fluorescent staining of actin.⁵⁵⁴ Cyanobacterial MC-LR was a potent and specific inhibitor of PP1 and PP2A from both mammals and higher plants.^554,565 MCs induce DNA damage in vitro and in vivo.(566) In HepG2 cells, MC-LR induced DNA strands break which were transiently present and likely produced during the cellular repair of MC-LR induced DNA damage.^566,567 The LD₅₀ by intraperitoneal injection (IP) of MC-LR was about 50 μg/kg bodyweight.⁵⁶⁸ MC-YR (544) were obtained from Microcystis aeruginosa strain CALU 972 collected in Lake Kroshnosero, Russia.^569,570 The absolute configuration of MC-YR was determined by amino acid analyses and fast atom bombardment mass spectrometry.⁵⁶⁹ MC-YR showed cytotoxicity, with EC₅₀ values of 35 μM for poeciliopsis lucida hepatocellular carcinoma 1 (PLHC-1) cells and 67 μM for the rainbow trout tissue (RTG-2) cell line.⁵⁷¹ Additionally, MC-YR resulted in the inhibition of neutral red uptake with reductions higher than 80% at 100 μM in undifferentiated Caco-2 cells after 48 h (EC₅₀ of 57.3 μM).⁵⁷² Chronic exposure to low doses of MC-YR (10 μg/kg, IP) may cause atrophy and fibrosis of the heart muscle.⁵⁷³ Chronic treatment of rats with intraperitoneal injections of sublethal doses of MC-LR (10 μg/kg) and MC-YR (10 μg/kg) induced liver and kidney injuries.⁵⁷⁴ MCs have a strong affinity to serine/threonine PPs, thereby acting as an inhibitor of this group of enzymes.⁵⁵⁴ Through this interaction a cascade of events responsible for the MCs’ cytotoxic and genotoxic effects in animal cells may take place.⁵⁷⁵ Moreover, MCs induced oxidative stress in animal cells and, together with the inhibition of PPs, this pathway was considered to be one of the main mechanisms of MC toxicity.⁵⁷⁵ MCs were shown to interact with the mitochondria.⁵⁷⁵ The consequences were the dysfunction of the organelle, induction of ROS, and cell apoptosis.⁵⁷⁵ MC activity led to the differential expression/activity of transcriptional factors and protein kinases involved in the pathways of cellular differentiation, proliferation, and tumor promotion activity.⁵⁷⁵ This activity may result from the direct inhibition of the PP1 and PP2A.⁵⁷⁵

MC peptides are produced nonribosomally via a multifunctional enzyme (a peptide synthetase).^576,577 Their synthesis is the result of an adenosine triphosphate (ATP)-dependent process.⁵⁷⁷ The enzymatic synthesiscomplex was codified by an mcy genes cluster composed of two operons (mcyA-C and mcyD-J),⁵⁷⁸ which was present in toxic strains of the genus Microcystis but also in microcystin-producing strains of Anabaena, Nostoc, and Anabaenopsis.⁵⁷⁷ Temperature has been shown to influence the type of toxin produced with high temperatures (>25 °C), enhancing MC-RR production and lower temperatures favoring MC-LR synthesis.⁵⁷⁹ The factors affecting the production of MCs include light, nitrogen, phosphorus, and so on.⁵⁷⁷

4.5.2. Aeruginosins

Aeroginosins are peptides that were first isolated by Murakami from the cyanobacterium Microcystis sp. in 1994.⁵⁸⁰ Aeruginosin 298-A (545)⁵⁸⁰ and 298-B (546)⁵⁸¹ were isolated from Microcystis aeruginosa (NIES-298) in Japan. The absolute configuration of aeruginosin 298-A and 298-B were initially determined by Marfey’s and chiral-phase HPLC methods.⁵⁸¹ The absolute configuration of aeruginosin 298-A was later revised through total synthesis.⁵⁸² Aeruginosin 298-A inhibited thrombin and trypsin with an IC₅₀ value of 0.3 and 1.0 μg/mL, respectively.⁵⁸⁰ The total synthesis of aeruginosin 298-A has been reported.⁵⁸² Aeruginosin 98-A (547),⁵⁸³ 98-B (548),⁵⁸³ and 98-C (549)⁵⁸¹ were isolated from Microcystis aeruginosa (NIES-98) in Japan. The absolute configurations of aeruginosin 98-A and 98-B were determined by the GC analysis on a chiral stationary phase of N-trifluoroacetyl isopropyl ester derivative of the acid hydrolysate.⁵⁸³ These were then compared to synthetic standards and ODS HPLC analyses of menthyl esters of the acid hydrolysates and Mosher’s method.⁵⁸¹ The absolute configuration of aeruginosin 98-C was determined by comparison with synthetic standards, Marfey’s, and chiral-phase HPLC methods.⁵⁸¹ The complete absolute configuration of aeruginosin 98-B was deduced by X-ray analysis.⁵⁸⁴ Aeruginosin 98-A inhibited trypsin with an IC₅₀ value of 0.6 μg/mL and plasmin and thrombin with IC₅₀ values of 6.0 and 7.0 μg/mL, respectively.^581,583 Aeruginosin 98-B also inhibited trypsin, plasmin, and thrombin with IC₅₀ values of 0.6, 7.0, and 10.0 μg/mL, respectively.^581,583 Aeruginosin 98-C inhibited trypsin, plasmin, and thrombin with IC₅₀ values of 3.9, 5.0, and 3.3 μg/mL, respectively.⁵⁸¹ The total synthesis of aeruginosin 98-B (548) has been reported.⁵⁸⁵ Aeruginosin 101 (550) was isolated from Microcystis aeruginosa (NIES-101) in Japan.⁵⁸¹ The absolute configuration of aeruginosin 101 was deduced by Marfey’s method, chiral-phase HPLC, and a chiral reagent.⁵⁸¹ Aeruginosin 101 inhibited trypsin with an IC₅₀ value of 3.0 μg/mL and plasmin and thrombin with IC₅₀ values of 3.3 and 3.2 μg/mL, respectively.⁵⁸¹ Aeruginosin 89-A (551) and 89-B (552) were isolated from Microcystis aeruginosa (NIES-89) in Japan.⁵⁸¹ The absolute configurations of aeruginosin 89-A and 89-B were deduced by Marfey’s and chiral-phase HPLC methods.⁵⁸¹ Aeruginosin 89-A inhibited trypsin, plasmin, and thrombin with IC₅₀ values of 0.4, 0.02, and 0.03 μg/mL, respectively.⁵⁸¹ Aeruginosin 89-B also inhibited trypsin, plasmin, and thrombin with an IC₅₀ value of 6.6, 0.46, and 0.05 μg/mL, respectively.⁵⁸¹ Aeruginosin 102-A (553) and 102-B (554) were isolated from Microcystis viridis (NIES-102) in Japan.⁵⁸⁶ The absolute configurations of aeruginosin 102-A and 102-B were determined by chiral-phase GC analysis of the N-trifluoroacetyl isopropyl ester derivative of the acid hydrolysate, Marfey’s method, and chiral-phase HPLC methods.⁵⁸⁶ Aeruginosin 102-A inhibited trypsin, thrombin, and plasmin with IC₅₀ values of 0.2, 0.04, and 0.3 μg/mL.^581,586 Aeruginosin 102-B also inhibited trypsin, thrombin, and plasmin with IC₅₀ values of 1.1, 0.1, and 0.8 μg/mL, respectively.⁵⁸⁶ Aeruginosin 103-A (555) was isolated from Microcystis viridis (NIES-103) in Japan.⁵⁸⁷ The absolute configuration of aeruginosin 103-A was deduced by HPLC analysis and Marfey’s method.⁵⁸⁷ Aeruginosin 103-A inhibited thrombin, trypsin, and plasmin with an IC₅₀ value of 9.0, 51.0, and 68.0 μg/mL, respectively.⁵⁸⁷ Aeruginosin 205-A (556) and 205-B (557) were isolated from Oscillatoria agardhii (NIES-205) in Kasumigaura Lake, Japan.⁵⁸⁸ The absolute configurations of aeruginosin 205-A and 205-B were determined by HPLC analysis of the menthyl ester derivatives of the acid hydrolysates, Marfey’s method, and chiral-phase GC analyses of the acid hydrolysates.⁵⁸⁸ The structure of aeruginosin 205-A was later revised, clarifying the position of the sulfate group.⁵⁸⁹ The structure of aeruginosin 205-B was revised after a stereoselective synthesis.⁵⁹⁰ Both aeruginosin 205-A and 205-B inhibited trypsin with an IC₅₀ value of 0.07 μg/mL.⁵⁸⁸ Aeruginosin 205-A and 205-B also inhibited thrombin with IC₅₀ values of 1.5 and 0.17 μg/mL, respectively.⁵⁸⁸ The total synthesis of aeruginosin 205-B has been reported.⁵⁹⁰ Aeruginosin KT608-A (558), KT608-B (559), and KT650 (560) were isolated from Microcystis aeruginosa in Lake Kinneret, Israel.⁵⁹¹ Advanced Marfey’s and chiral-phase HPLC methods were used to determine the absolute configuration of aeruginosin KT608-A-C.⁵⁹¹ The absolute configuration of aeruginosin KT608-A was revised following a stereoselective synthesis in 2017.⁵⁹² Aeruginosin KT608-A-C inhibited trypsin with IC₅₀ values of 1.9, 1.3, 19.9 μM, respectively.⁵⁹¹ The total synthesis of aeruginosin KT608-A (558) has been reported.⁵⁹² Aeruginosin EI461 (561) was isolated from Microcystis aeruginosa in the Einan Reservoir in the Hula Valley, Israel.⁵⁹³ Marfey’s and the (−)-menthol methods were used to determine the absolute configuration of aeruginosin EI461.⁵⁹³ The absolute configuration of aeruginosin EI461 was later revised following a stereoselective synthesis in 2003.⁵⁹⁴ Aeruginosin EI461 inhibited 15% of the activity of trypsin at a concentration of 45.5 μg/mL.⁵⁹³ The total synthesis of aeruginosin EI461 (561) has been reported.⁵⁹⁴ Aeruginosin KB676 (562) was isolated from Microcystis spp. in Kibbutz Kfar Blum, Israel.⁵⁹⁵ Marfey’s and chiral-phase HPLC methods were used to determine the absolute configuration of aeruginosin KB676.⁵⁹⁵ Aeruginosin KB676 inhibited trypsin and chymotrypsin with IC₅₀ values of 40 and >45.5 μM, respectively.⁵⁹⁵ Aeruginosin 828-A (563) was isolated from Planktothrix in Switzerland.⁵⁹⁶ The relative configuration of aeruginosin 828-A was established by NOESY and J-value analysis.⁵⁹⁶ Additionally, very potent inhibition values for thrombin (IC₅₀ = 21.8 nM) and trypsin (IC₅₀ = 112 nM) have been measured for aeruginosin 828-A.⁵⁹⁶ Aeruginosin 828-A was found to be toxic to Thamnocephalus platyurus with an LC₅₀ value of 22.4 μM.⁵⁹⁶ The total synthesis of aeruginosin 828-A (563) has been reported.⁵⁹⁷ Aeruginosin 126-A (564) and 126-B (565) were isolated from Planktothrix agardhii CYA126/8 in Finland.⁵⁹⁸ The absolute configuration of aeruginosin 126-A was determined following chromatographic analysis of the acid hydrolysate on a chiral stationary phase and comparison of its retention time to those of authentic standards.⁵⁹⁸ Aeruginosin 126-A weakly inhibited porcine pancreas trypsin and bovine plasma thrombin with IC₅₀ values of 67 and 30 μg/mL, respectively.⁵⁹⁸ The total synthesis of aeruginosin 126-A (564) has been reported.⁵⁹⁷ Aeruginosin 865 (566) was isolated from Nostoc sp. in Krusne mountains, Czech Republic, and chiral-phase HPLC analysis of Marfey derivatives was applied to determine the peptide sequence.⁵⁹⁹ Aeruginosin 865 inhibited IL-8 and intercellular cell adhesion molecule 1 (ICAM-1) in human TNF-α-stimulated human lung microvascular endothelial cells (HLMVECs), with EC₅₀ values of 3.5 ± 1.5 and 50.0 ± 13.4 μg/mL, respectively.⁵⁹⁹ Aeruginosin GH553 (567) was isolated from Microcystis spp. in Kibbutz Giva’at Haim, Israel.⁵⁹¹ Marfey’s analysis using l-fluorescent d-amino acid (FDAA) and d-FDAA as the coupling reagents established the d-configuration of tyrosine (Tyr) and diepi-2-carboxy-6-hydroxyoctahydroindole (Choi).⁵⁹¹ Analysis of the p-hydroxyphenyllactic acid (Hpla) on a chiral-phase HPLC column established its absolute configuration as l.⁵⁹¹ Aeruginosin GH553 inhibited trypsin with an IC₅₀ value of 45.5 μM, but not thrombin at the same concentration.⁵⁹¹

4.5.2.1. Biosynthesis of Aeruginosins

Aeruginosins were synthesized on nonribosomal peptide synthetase (NRPS) enzyme complexes encoded in the aeruginosin (aer) biosynthetic gene cluster (BGC).^598,600,601 Aeruginosin biosynthesis begins with the activation of a monocarboxylic acid or fatty acid by the loading module aerA.^598,601,602AerB was responsible for adding a hydrophobic d-amino acid.^598,601AerD, aerE, and aerF worked in a cascading manner to synthesize and supply Choi to aerG.^598,602AerG added a Choi moiety and encoded an off-loading module.^598,601 Variation in the loading and off-loading mechanisms of aeruginosin synthetases contributes to the observed structural diversity of the natural product family.⁶⁰² Aeruginosin BGCs also encoded an assortment of tailoring enzymes that promoted structural diversification, including halogenation, acylation, and glycosylation.⁶⁰²

4.5.3. Cryptophycins

Cryptophycins are 16-membered cyclic depsipeptides which are potent (picomolar) tumor-selective cytotoxins associated with the terrestrial blue-green algae Nostoc sp. that have shown promising anticancer properties. Researchers at Merck initially isolated cryptophycin-1 (568) in 1990 from Nostoc sp. ATCC 53789 with potent activity against the fungus Cryptococcus neoformans but decided not to pursue it clinically due to its toxicity in mice resulting in a low therapeutic index.⁶⁰³ In 1993, Moore’s group at the University of Hawaii isolated cryptophycin-1 from Nostoc sp. GSV 224 and designated it cryptophycin A together with minor analogs (B-G) from their large blue-green algae screening program.^604,605 Eighteen minor cryptophycin compounds were described along with their structure elucidation, including X-ray analysis, Marfey analysis of acid hydrolysates, conformational analysis, chemical stability studies, and antitumor activity.⁶⁰⁶ Additional cryptophycins were isolated from terrestrial Nostoc in 2004.⁶⁰⁷

Cryptophycin A demonstrated in vitro tumor cell selectivity in the Corbett/Valeriote assay;⁶⁰⁸ and, in vivo therapeutic efficacy against innately resistant murine PANC03 as well as both sensitive and Taxol-resistant Mammary 16/C tumors.⁶⁰⁵ Subsequently many syngeneic murine as well as human xenograft tumors have shown excellent sensitivity to cryptophycins and also cryptophycin combinations with other standard anticancer drugs and radiation.⁶⁰⁹ The mechanism of action of cryptophycin was quickly defined as inhibiting tubulin assembly into microtubules by binding at the same site as the Vinca alkaloids.^610,611 Also, bcl phosphorylation is induced leading to apoptosis. Importantly, cryptophycin was equally active against P-glycoprotein-resistant cancer cells.⁶¹⁰

Preclinical studies of cryptophycin-8 (574) were evaluated for activity against subcutaneous tumors of both mouse and human origin. Cryptophycin-8 was less potent than cryptophycin-1 by approximately 4-fold; however, it was both more water-soluble and had greater therapeutic efficacy.⁶¹²

The first total synthesis of cryptophycins was a convergent synthesis of four separate fragments (units A–D) coupled to form the final product yielding cryptophycins C and D.²⁵⁹ The synthesis of cryptophycins C and D showed that both natural products had the D-tyrosine α-amino acid (Figure 7). The study also revised the structures of cryptophycins A and C to show the D-configuration of the α-amino acid unit.²⁵⁹ Many natural and synthetic analogues of cryptophycin A were prepared by the Moore lab but none were more efficacious than cryptophycin A.^606,613 The C-6 gem-dimethyl analogue, cryptophycin-52 (573), was chosen from over 450 cryptophycin analogues and had all of the positive biological attributes of cryptophycin A but allowed for a more efficient synthesis and possessed stability of the ester bond between Units C and D to become the candidate for clinical trial.⁶¹⁴

Figure 7.

Numbering system for each unit of cryptophycins C (R₁=C1) and D (R₁=H).

Lilly Pharmaceuticals supported Phase 1 clinical trials for cryptophycin-52 (LY355703, 573) which suggested that the drug be administered on days 1 and 8 on a 21 day schedule at 1.48 mg/m² for a Phase 2 trial.^615,616 Peripheral neuropathy and myalgia, similar to that noted for other tubulin inhibitors, appeared to be the main concern. This schedule was employed in a Phase 2 study for patients with advanced non-small cell lung cancer previously treated with a platinum.⁶¹⁷ While 40% of patients showed disease stabilization, the dose had to be reduced and neurotoxicity was dose limiting. In another Phase 2 study on platinum-resistant ovarian cancer, the same schedule with 1.5 mg/m² demonstrated overall clinical benefit with 41.7% partial response or stable disease with the drug well tolerated.⁶¹⁸

Both regular and chemoenzymatic syntheses has been developed in an attempt to prepare more effective cryptophycin analogues and a wide synthetic literature exists.⁶¹³ These include the chlorohydrin analog cryptophycin-8,⁶¹² glycinated analogs,⁶¹⁹ A-unit modifications,⁶²⁰ and antibody-drug conjugates.^621,622 For further studies on the cryptophycins, note the following reviews.^623−626

4.5.4. Others

Anabaenopeptins KB906 (575) and KB899 (576) were isolated from Microcystis spp. in Kfar Blum, Jordan Valley, Israel.⁵⁹⁵ Marfey’s method was used to determine the absolute configuration of the molecules.⁵⁹⁵ Anabaenopeptin KB906 and KB899 inhibited trypsin and chymotrypsin with IC₅₀ values both >45.5 μM.⁵⁹⁵ Microphycin KB921 (577), KB928 (578), KB956 (579), KB970A (580), KB970B (581), KB984 (582), KB970C (583), KB1048 (584), KB992 (585), and KB1046 (586) were isolated from Microcystis spp. in Kfar Blum, Jordan Valley, Israel.⁵⁹⁵ Both Marfey’s method and chiral-phase HPLC analysis were used to determine the absolute configuration of the compounds.⁵⁹⁵ KB928, 956, 970A, 970B, 984, 970C, 1048 inhibited trypsin with IC₅₀ values of 0.09, 0.62, 0.09, 0.65, 1.12, 4.27, 2.01 μM, respectively. KB1048, 992, and 1046 inhibited chymotrypsin with IC₅₀ values of 0.63, 0.87, and 0.22 μM, respectively.⁵⁹⁵ KB921, 928, 956, 970A, 970B, 984, 970C inhibited chymotrypsin with IC₅₀ values all of >45.5 μM.⁵⁹⁵ Micropeptin KT1042 (587), KT636 (588), and pseudoaeruginosin KT554 (589) were isolated from Microcystis aeruginosa in Lake Kinneret, Israel.⁵⁹¹ The absolute configurations of KT1042 and KT554 were determined by Marfey’s analysis and chiral-phase HPLC methods.⁵⁹¹ KT1042 inhibited chymotrypsin with an IC₅₀ value of 0.26 μM, but it did not inhibit trypsin, thrombin, or elastase at a concentration of 45.5 μM.⁵⁹¹

4.6. Amino Acids

4.6.1. Kainoids

Domoic acid (DA, 590) was isolated from Pseudonitzschia sp. in Cardigan Bay, Canada⁶²⁷ and Amphora sp. in Prince Edward Island, Canada.^628,629 The absolute configuration of DA was determined by X-ray crystal analysis and revised through total synthesis.⁶³⁰ The LD₅₀ of DA was 6.0 mg/kg with a 95% confidence interval between 4.2 and 7.9 mg/kg.⁶³¹ DA significantly decreased the number of dopaminergic neurons (LC₅₀ = 100 μM), decreased the expression of neuronal nuclear antigen, and slightly affected astrocyte populations. In addition, compound 590 increased the release of lactate dehydrogenase into the culture media.⁶³² 2′,7′-Dichlorodihydrofluorescein diacetate (H₂DCFDA), JC-1, and 4′,6-diamidino-2-phenylindole (DAPI) fluorescence staining, respectively, revealed that DA slightly raised ROS production and significantly decreased mitochondrial membrane potential and increased apoptotic cell death of cultured cells, significantly reduced the number of dopaminergic neurons by about 15 and 46% at the concentrations 10 μM and 100 μM, respectively.⁶³² DA was able to reach cardiac tissue after IP (2.5 mg/kg) exposition and was able to traverse the cell membrane in rats. Transmission electron microscopy (TEM) showed the loss of myofibrillar arrangement and severe mitochondrial alterations in the heart.⁶³³ The total synthesis of DA has been reported.^630,634 Kainic acid (KA, 591) was isolated from Digenea simplex in Japan.⁶³⁵ The absolute configuration of KA was determined by X-ray crystal analysis.⁶³⁵ KA causes the fast degeneration of neurons within cell bodies within the area of injection (2 μg, rat striatum)⁶³⁶ and induces apoptosis in neurons (100 μM).⁶³⁷ KA (1.67 μg/kg) had its greatest destructive action on neuronal perikarya, a significant amount of damage to the axons of passage may also occur.⁶³⁸ The total synthesis of KA has been reported.^639−642 Allokainic acid (592) was isolated from Digenea simplex in Japan.⁶³⁵ The absolute configuration of allokainic acid was determined by X-ray crystal analysis.^635,643 Isodomoic acids A (593), B (594), and C (595) were isolated from Chondria armata in Japan.⁶⁴⁴ The relative configurations of isodomoic acids A, B, and C were determined by NOE data analysis.⁶⁴⁴ Isodomoic acids A, B, and C had insecticidal effects on American cockroaches with a minimum effective dose (MED) of 32, 32, and 64 nmol, respectively.⁶⁴⁴ The total syntheses of (−)-isodomoic acids B (594)⁶⁴⁵ and isodomoic acids C (595)⁶⁴⁶ have been reported. Both the 7′-methylisodomoic acids (A, 596 and B, 597) and 7′-hydroxymethylisodomoic acids (A, 598 and B, 599) were isolated from Chondria armata in Kagoshima prefecture, Japan.⁶⁴⁷ The relative configurations of 7′-methyl-isodomoic acids A and 7′-hydroxymethyl-isodomoic acids B were determined by NOESY analysis.⁶⁴⁷ The absolute configurations of compounds 7′-methyl-isodomoic acids A and 7′-hydroxymethyl-isodomoic acids B were established through the comparison of NMR data with DA.⁶⁴⁷ Isodomoic acid D (600) was isolated from Chondria armata.(648) Isodomoic acids E (601) and F (602) were isolated from laboratory cultures ofNitzschia pungens.⁶⁴⁸ The relative configurations of isodomoic acids D, E, and F were determined by the analysis of NOE experiments.⁶⁴⁸ Isodomoic acid D (IC₅₀ = 53 μM), isodomoic acid E (IC₅₀ = 300 μM), and isodomoic acid F (IC₅₀ = 14 μM) all exhibited binding to a population of low-affinity α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) sites.⁶⁴⁹ Intrahippocampal injection of isodomoic acid D (ED₅₀ = 3317 pmol), isodomoic acid E (ED₅₀ = 4000 pmol), and isodomoic acid F (ED₅₀ = 368 pmol) all produced dose- and time-dependent increases in seizure activity.⁶⁴⁹ The total synthesis of (−)-isodomoic acid E and (−)-isodomoic acid F have been reported.⁶⁴⁵ Isodomoic acid G (603) and H (604) were isolated from Chondria armata in Kyushu Island, Japan.⁶⁵⁰ The relative configuration of isodomoic acid G and isodomoic acid H were determined by NOESY analysis.⁶⁵⁰ The absolute configuration of isodomoic acid G was determined through a stereoselective total synthesis.^651,652 Additionally, the total synthesis of isodomoic acid H has been reported.⁶⁵² Domoilactone A (605) and B (606) were isolated from Chondria armata in Japan.⁶⁴⁴ The absolute configuration of domoilactone A and domoilactone B was inferred on the basis of the NOE experiments and biogenetic considerations.⁶⁴⁴

4.6.2. Biosynthesis of Domoic Acid

By using whole genome sequencing, genes homologous to both dabA and dabC were discovered from two KA-producing red algae, Digenea simplex, and Palmaria palmata, and named kabA and kabC.⁶⁵³ Heterologous expression of kabA and kabC in Escherichia coli and subsequent in vitro activity assays demonstrated that kabA catalyzes the N-prenylation of l-Glu using dimethylallyl diphosphate (DA) as a prenyl donor to yield the intermediate prekainic acid.⁶⁵³ KabC, an αKG-dependent dioxygenase, then cyclizes prekainic acid to generate KA.⁶⁵⁴ In 2012, it was suggested that DA is synthesized through the condensation of l-glutamic acid and geranyl diphosphate via anucleophilic displacement reaction, supported by the experimental incorporation of [1-²H₂] geraniol into DA using Pseudonitzschia spp.⁶⁵⁵ Subsequently, Yamashita et al. (2018) identified six new potential biosynthetic intermediates of DA and put forth a biosynthetic scheme for DA.⁶⁴⁷

4.6.3. β-N-Methylamino-l-alanine

β-N-Methylamino-l-alanine (BMAA, 607), a nonproteinogenic amino acid, was found to be produced by cyanobacterial root symbionts of the genus Nostoc.(656) BMAA was suggested as a possible cause of the amyotrophic lateral sclerosis/parkinsonism–dementia (ALS/PD) that has an extremely high rate of incidence among the Chamorro people of Guam compared with incidence rates of ALS elsewhere.⁶⁵⁷

5. Therapeutics

5.1. Cytotoxicity

Phytoplankton toxins demonstrate cytotoxic effects on various cancer cell lines.^92,658 The ability of phytoplankton toxins to halt the growth of cancer cell lines indicates the potential for the development of potent anticancer drugs.⁶⁵⁹ Some phytoplankton toxins directly act on cancer cell lines through cytotoxicity^{92,385,389,658} (Table 1). Amphidinolides exhibited robust in vitro cytotoxicity (Table 1).⁶⁵⁸ Out of all the amphidinolides, amphidinolide N (25) demonstrated the most potent antitumor activity, with IC₅₀ values of 0.05 ng/mL for L1210 cells and 0.06 ng/mL for KB cells,⁹² with a preference for malignant cells’ mitochondria. Amphidinolide H (14) appears to target the actin cytoskeleton.⁶⁶⁰ BSXs exhibit cytotoxic effects on neuroblastoma cells (Table 1).^385,389 Moreover, it exerts a dose-dependent impact on cell growth, induces cell death through apoptosis, and possesses genotoxic properties in Jurkat E6–1 cells and leukemic T-cell lines.^661,662 PTXs and analogs are strongly cytotoxic against various human cancer cell lines (Table 1).⁶⁶³ PTX-2 (532), an actin inhibitor, has been suggested as a potential chemotherapeutic treatment for malignancies lacking functional p53.⁶⁶⁴ Additionally, AMs,¹⁰⁹ lingshuiols,¹³⁹ KmTx 8 (138),¹⁵⁴ ostreol B (142),¹⁵⁹ 4-hydroxyprorocentrolide (146),¹⁶⁶ prorocentrolide C (147),¹⁶⁶ lyngbyatoxins,³²⁴ pinnatoxin D (296),³⁵⁵ KBTs,^386−388 gymnocins,^467,468,470 prorocentin (454), belizeanolide (494) and belizeanolic acid (495),^510,515 limaol (497),⁵¹⁷ neo-debromoaplysiatoxin J (167),¹⁹¹ aplysiaenal (172)¹⁹⁴ and OTXs¹⁹⁸ all displayed cytotoxic effects on many types of cancer cells (Table 1).

Table 1. Cytotoxicity of Phytoplankton Toxins.

Cell lines	Compound no.	IC50/EC50/GI50	Ref
L1210	1	2.4 μg/mL	(51)
	5	0.00012 μg/mL	(53)
	6	0.0014 μg/mL	(53)
	9	5.8 ng/mL	(55)
	10	19 ng/mL	(56)
	11	2.0 μg/mL	(57)
	12	1.5 μg/mL	(58)
	13	5.4 ng/mL	(59)
	14	0.48 ng/mL	(59)
	15	0.3 μg/mL	(60)
	16	0.72 μg/mL	(60)
	17	0.06 μg/mL	(60)
	18	0.002 μg/mL	(60)
	19	0.18 μg/mL	(60)
	20	0.2 μg/mL	(60)
	21	2.7 μg/mL	(61)
	22	1.65 μg/mL	(62)
	23	0.092 μg/mL	(63)
	24	1.1 μg/mL	(64)
	25	0.05 ng/mL	(92)
	26	1.7 μg/mL	(65, 95)
	27	1.6 μg/mL	(65, 95)
	28	6.4 μg/mL	(66)
	29	1.4 μg/mL	(67)
	30	4.0 μg/mL	(67)
	31	18 μg/mL	(68)
	32	15 μg/mL	(68, 69, 102)
	33	10 μg/mL	(68, 69, 102)
	34	7 μg/mL	(68, 69, 102)
	35	11 μg/mL	(68, 69, 102)
	36	12 μg/mL	(70)
	37	3.2 μg/mL	(71)
	38	3.9 μg/mL	(72)
	39	0.6 μg/mL	(73)
	40	0.8 μg/mL	(74)
	463	48 μM	(198)
	464	52 μM	(198)
	466	29 μM	(198)
	265	8.1 μM	(324)
	266	20.4 μM	(324)
L5178Y	1	3.9 μg/mL	(51)
	11	4.8 μg/mL	(57)
HCT 116	2	0.122 μg/mL	(52)
	3	7.5 μg/mL	(52)
	4	0.206 μg/mL	(52)
	142	0.9 μM	(159)
	147	2.2 μM	(165)
	497	7.3 μM	(517)
KB	5	0.001 μg/mL	(53)
	6	0.004 μg/mL	(53)
	12	3.2 μg/mL	(58)
	13	5.9 ng/mL	(59)
	14	0.52 ng/mL	(59)
	15	0.8 μg/mL	(60)
	16	1.3 μg/mL	(60)
	17	0.06 μg/mL	(60)
	18	0.022 μg/mL	(60)
	19	0.23 μg/mL	(60)
	20	0.6 μg/mL	(60)
	21	3.9 μg/mL	(61)
	22	2.9 μg/mL	(62)
	23	0.1 μg/mL	(63)
	24	0.44 μg/mL	(64)
	25	0.06 ng/mL	(92)
	26	3.6 μg/mL	(65, 95)
	27	5.8 μg/mL	(65, 95)
	29	0.67 μg/mL	(67)
	30	6.5 μg/mL	(67)
	31	35 μg/mL	(68, 69, 102)
	32	20 μg/mL	(68, 69, 102)
	33	11.5 μg/mL	(68, 69, 102)
	34	10 μg/mL	(68, 69, 102)
	35	18 μg/mL	(68, 69, 102)
	36	20 μg/mL	(70)
	37	7 μg/mL	(71)
	39	7.5 μg/mL	(73)
	40	8.0 μg/mL	(74)
DG-75	7	0.02 μg/mL	(54)
	8	0.4 μg/mL	(54)
P388	47	25.3 μg/mL	(117)
	48	36.5 μg/mL	(117)
	49	35.2 μg/mL	(117)
	50	23.0 μg/mL	(117)
	51	26.8 μg/mL	(117)
	52	32.5 μg/mL	(117)
	296	2.5 μg/mL	(355)
	348	10.9 nM	(386)
	349	3.5 nM	(386)
	350	2.7 nM	(387)
	351	0.7 nM	(388)
	352	1.6 nM	(388)
	353	0.14 nM	(388)
	386	1.3 μg/mL	(467, 468)
	387	1.7 μg/mL	(470)
A549	60	8 μM	(109)
	122	0.21 μM	(137)
	146	17.8 μM	(166)
	147	14.6 μM	(166)
A2058	60	16.4 μM	(109)
HepG2	60	6.8 μM	(109)
	142	4.8 μM	(159)
	497	3.7 μM	(517)
MCF7	60	16.8 μM	(109)
MiaPaCa-2	60	8.6 μM	(109)
HL-60	122	0.23 μM	(137)
SR	138	0.100 μM	(154)
CCRF-CEM	138	0.686 μM	(154)
HOP-62	138	0.986 μM	(154)
NCI-H23	138	0.903 μM	(154)
HOP-92	138	0.501 μM	(154)
IGROV1	138	0.631 μM	(154)
SN12C	138	1.000 μM	(154)
BT-549	138	0.501 μM	(154)
HeLa	138	1064 nM	(154)
	265	35 μM	(324)
Neuro-2a	142	0.1 μM	(159)
	147	5.2 μM	(165)
	354	300 ng/mL	(389)
	355	370 nM	(385)
	356	20 ng/mL	(389)
	357	26.9 nM	(385)
	497	9.6 μM	(517)
HT-29	146	9.9 μM	(166)
	147	10.5 μM	(166)
ACC-MESO-1	265	11 μM	(324)
DLD-1	454	16.7 μg/mL	(510)
RPMI7951	454	83.6 μg/mL	(510)
A2780	494	3.28 ± 0.45 μM	(515)
	495	0.26 ± 0.09 μM	(515)
SW1573	494	3.23 ± 0.45 μM	(515)
	495	0.31 ± 0.06 μM	(515)
T47D	494	3.16 ± 0.40 μM	(515)
	495	0.40 ± 0.09 μM	(515)
WiDr	494	4.58 ± 0.40 μM	(515)
	495	0.41 ± 0.04 μM	(515)
RD	532	23 ng/mL	(529)
SW480	167	4.63 ± 0.20 μM	(191)

Other phytoplankton toxins indirectly act on cancer cell lines by inducing cell death, synergistic actions, or causing cellular damage. By inducing cell death through cholesterol depletion, KmTx could be developed as a novel chemotherapeutic drug for managing cancer across a range of solid tumor lines, including prostate and breast cancer cells.^154,659,665 The synergistic effects of gymnodimine (A) (258) (10 μM) and its analogs, along with OA (434) (100 nM), could be utilized therapeutically to enhance anticancer efficacy by inducing toxicity in tumor cells and serving as chemotherapeutic agents.⁶⁶⁶ YTX (538) also induced nonapoptotic cell death in the BE (2)-M17 neuroblastoma cell line (1000 nM),⁶⁶⁷ L6 and BC3H1 myoblast cells (100 nM),⁶⁶⁸ and glioma cells (30 nM and 250 nM).⁵³⁷ Recent findings have revealed that it induces mitotic catastrophe and genetic alterations at a concentration of 100 nM, suggesting its potential utility in managing cancer progression.^659,669 The spiroimine, portimine A (324), has promising low nanomolar activity in vitro and efficacy in in vivo models as well.³⁶² MCs are resilient hydrophilic cyclic heptapeptides capable of inducing cellular damage upon uptake through organic anion-transporting polypeptides (OATPs).^670,671 Owing to the overexpression of certain OATPs in tumors relative to normal tissues, microcystins represent promising targets for the development of anticancer drugs.^659,670 Cancer cells possess elevated levels of intrinsic oxidative stress, which renders them susceptible to external assaults from ROS.⁶⁷¹ Consequently, analogs of microcystin can selectively eliminate cancer cells expressing OATP while causing minimal harm to healthy cells.⁶⁷¹ In summary, phytoplankton toxins represent a promising approach to circumvent cancer treatment delays or obstacles.⁶⁵⁹

5.2. Antifungal

The increase in antifungal drug resistance is a major global human health concern, particularly given the limited number of antifungal drugs available to treat invasive infections.⁶⁷² Some phytoplankton toxins exhibited antifungal activity (Table 2), making them potential starting points for antifungal drug discovery.^{133,136,659,673} AMs (Table 2) exhibited a higher affinity for the ergosterol membrane, suggesting the formation of a more stable complex. This observation could pave the way for the development of novel antifungal drugs.^44,673 Carteraol E (116) (15 μg/disk)¹³³ and karatungiol A (120) (12 μg/disk)¹³⁶ showed strong antifungal activity against Aspergillus niger (Table 2). Luteophanol A (125) exhibited weak antimicrobial activity against Sarcina lutea (MIC = 33 μg/mL) (Table 2).¹⁴⁰ The topic of antifungals from cyanobacteria was recently comprehensively reviewed by S.C. do Amaral et al. and published in Marine Drugs in 2023.⁶⁷⁴

Table 2. Antifungal Activity of Phytoplankton Toxins.

	MIC/MEC
Compound no.	Aspergillus niger (μg/disk)	Candida albicans (μg/mL)	Sarcina lutea (μg/mL)	Aspergillus flavus (μg/mL)	Ref
41	6				(115)
42	6				(108)
43	9.				(44)
44	6				(115)
45	6				(115)
46	10				(116)
47	58.2				(117)
48	32.9				(117)
56		9			(120)
64		19			(123)
66		16		4	(124)
116	15				(133)
120	12				(136)
125			33		(140)

5.3. Antibacterial

Luteophanol A (125) exhibited weak antimicrobial activity against Gram-positive bacteria (MIC values: Staphylococcus aureus, 33 μg/mL; Bacillus subtilis, 66 μg/mL).¹⁴⁰ Luteophanol D (128) exhibited an antibacterial activity against Micrococcus luteus (MIC, 33 μg/mL).¹⁴²

5.4. Antiprotozoan

Karatungiol A (120) exhibited antiprotozoan activity against Trichomonas fetus at 1 μg/mL.¹³⁶

5.5. Artemia Toxicity

Ostreol A (141) exhibited cytotoxicity against Artemia salina with an IC₅₀ value of 0.9 μg/mL.⁶⁷⁵

5.6. Analgesic

NEOSTX (235) functions as a prolonged, local pain blocker.²⁸⁶ Five patients with bladder pain syndrome (BPS) received a total dose of 80 μg of NEOSTX in an isoosmotic solution of 0.9% NaCl, pH 6.5.²⁸⁶ The infiltration of NEOSTX was well tolerated by the patients, with pain blockage and beneficial effects lasting over the 90 days of observation. After the initial infiltration procedure, no second treatment was applied.²⁸⁶ The efficacy of this therapeutic approach is higher than any other conventional treatment known to date.²⁸⁶ GTX 2 (240) and 3 (241) have also been utilized in the treatment of chronic tension-type headaches.⁶⁷⁶ Twenty-seven patients diagnosed with chronic tension-type headache underwent local infiltration of GTX 2 and 3 (50 μg) at 10 identified pain trigger points.⁶⁷⁶ Two hundred microliters were injected at each designated site.⁶⁷⁶ No adverse effects were identified during the follow-up period.⁶⁷⁶ Nineteen out of the 27 patients (70%) responded successfully to the treatment.⁶⁷⁶ The safe and effective therapeutic properties of local infiltration with GTXs in patients with chronic tension-type headaches have been reported.⁶⁷⁶ GTXs are also employed as a pain alleviator in both acute and chronic anal fissures.⁶⁷⁷ One milliliter containing 100 units of gonyautoxin (238) was injected into both sides of the anal fissure within the internal anal sphincter.⁶⁷⁷ Complete resolution of acute anal fissures was attained within 15 days, while chronic anal fissures achieved total remission within 28 days.⁶⁷⁷ Ninety-eight percent of the patients experienced healing before 28 days, with an average healing time of 17.6 ± 9 days.⁶⁷⁷ No cases of fecal incontinence or other side effects were noted.⁶⁷⁷ GTXs disrupt the harmful cycle of pain and spasm that contributes to the development of anal fissures.⁶⁷⁷ The infiltration of GTXs into the anal sphincter represents a secure and effective alternative therapeutic approach compared to conservative, surgical, and botulinum toxin therapies for anal fissures.⁶⁷⁷

5.7. Alzheimer’s Disease (AD)

Recent discoveries have implicated soluble forms of Aβ and tau as the principal toxic agents in AD.⁶⁷⁸ YTX (538) and its analogs may have potential applications in the treatment of AD by reducing the levels of tau and Aβ.^537,547,679 At a concentration of 1 nM, YTX activates PKC with advantageous effects on the principal hallmarks of AD, namely tau and Aβ, in a cellular model derived from fetuses of triple transgenic mice (3xTg)-AD.⁵⁴⁷ Gymnodimine (A) (258) may contribute to reducing levels of amyloid and phosphorylation of tau.³¹⁵ Treating cortical neurons with a concentration of 50 nM of gymnodimine (A) resulted in a reduction in intracellular accumulation of Aβ and lowered levels of hyperphosphorylated isoforms of tau protein, as identified by AT8 and AT100 antibodies.³¹⁵ Spirolides have also been shown to play a neuroprotective role in AD.⁶⁸⁰ Treating 3xTg cortical neurons in vitro with 13-demethylspirolide C (286) at a concentration of 50 nM resulted in a decrease in intracellular accumulation of Aβ and reduced levels of phosphorylated tau.⁶⁸⁰ Further research and development could help to treat degenerative illnesses.

5.8. Cardiovascular Diseases (CVD)

VCAM-1 is a protein that belongs to the immunoglobulin (Ig) superfamily.⁶⁸¹ VCAM-1 has emerged as a promising drug target for treating atherosclerosis and associated cardiovascular disease (CVD) due to its involvement in monocyte recruitment and selective up-regulation on atheroprone regions of the vascular endothelium.⁶⁸² Gibbosol A (117) displayed remarkable activation effects on VCAM-1 expression (64.2 μM), whereas gibbosol B (118) exhibited marked inhibitory activity on VCAM-1 expression, particularly at a concentration of 100.0 μg/mL (62.7 μM).¹³⁴ The addition of symbiopolyol (143) prior to TNF-R stimulation markedly decreased VCAM-1 expression in HUVECs with an IC₅₀ value of 8.23 μg/mL (6.62 μM).¹⁶⁰ At a concentration of 2 μM, ZT-A (149) induced aggregation in rabbit platelets that were dependent on TXA₂ and sensitive to genistein.¹⁷⁵ ZT-B (150) induced a contraction in a concentration-dependent manner in the isolated rabbit aorta with effective concentrations ranging from 10^–7 to 10^–5 M.¹⁷⁶

5.9. Central Nervous System

Oleamide (333) (10 and 20 mg/kg)³⁷⁹ and linoleamide (336) (EC₅₀ of 20 μM)³⁸² are sleep-inducing lipids that decrease body temperature and locomotor activity in a dose-dependent manner when administered to rats. Erucamide (334) (5–20 mg/kg) administered orally may alleviate depression- and anxiety-like behaviors in mice.³⁸⁰ These activities may give phytoplankton toxins the potential to become sedative-hypnotic drugs. At a concentration of 30 nM, BTX B (360) demonstrated neuro-activation properties and has the potential to enhance neuronal plasticity. This quality could be valuable in pharmaceutical treatments aimed at restoring brain function after a stroke or other traumatic brain injuries.⁶⁸³

5.10. Antiviral

CHIKV is induced by an alphavirus and is transmitted to humans through mosquitoes of the Aedes species.⁶⁸⁴ Debromoaplysiatoxin (152) and 3-methoxydebromoaplysiatoxin (154) demonstrated noteworthy effectiveness against CHIKV with EC₅₀ values of 1.3 and 2.7 μM, respectively. They exhibited selectivity indices of 10.9 and 9.2, respectively.¹⁸²

5.11. Anti-inflammatory and Immunomodulatory Effects

The anti-inflammatory and immunomodulatory effects of OA (434) and YTX (538) suggest their potential immunomodulatory impact. They downregulate T-cell receptor expression, influencing T-cell function in immune responsiveness, and consequently affect the overall immunological response.^659,685 The downregulation of the T cell receptor complex (TCR) induced by YTX at a concentration of 25 nM was partially mediated through the activation of PKC.⁶⁸⁵ In contrast, the downregulation of the TCR induced by OA at a concentration of 100 nM was mediated by the inhibition of the serine/threonine phosphatase PP2A.⁶⁸⁵ A concentration of 100 nM of OA can also induce an inflammatory response in HL-60 human cells by significantly elevating the levels of IL-8.⁶⁸⁶

5.12. Insecticide

Isodomoic acids A (593), B (594), and C (595) had insecticidal effects on American cockroaches with MED of 32, 32, and 64 nmol, respectively.⁶⁴⁴

5.13. Sodium Channel Inhibitors

Neosaxitoxin (235) (10 nM or 0.2 nmol/day, 28 days) blocks neuronal voltage-dependent Na⁺ channels and could be an innovative drug to block the PCO phenotype, permitting the rats to ovulate and likely recover from decreased fertilization capacity that occurs after a sympathetic discharge, such as chronic sympathetic stress.²⁸⁴ Neosaxitoxin may be used as a treatment method for PCOS.²⁸⁴ Saxitoxin (232) predominantly inhibits sodium channels in both nerve and muscle cells⁶⁸⁷ leading to a state of paralysis.⁶⁸⁸ They also serve as potential therapeutics, functioning as anesthetic agents.^689,690 The median concentrations for neosaxitoxin and saxitoxin required to achieve a 60 min duration of analgesia were 34 ± 2 μmol/L and 58 ± 3 μmol/L, respectively.⁶⁹¹

5.14. Potassium Channel Inhibitors

ATXs and OTXs were the most representative structures with potassium channel inhibitory activities selectively against Kv1.5 (Table 3).^{186,188−191,202} Kv1.5 is considered a crucial target for the development of new treatments for atrial tachyarrhythmias with minimal side effects.¹⁸⁸ Researchers seeking novel approaches to treat atrial tachyarrhythmias may find these findings valuable.^188,659

Table 3. Potassium Channel Inhibitory Activities of ATXs and OTXs.

Compound no.	IC50 (μM)	Ref
158	6.94 ± 0.26	(186)
159	0.30 ± 0.05	(186)
162	1.22 ± 0.22	(189)
163	2.85 ± 0.29	(189)
164	1.79 ± 0.22	(190)
165	1.46 ± 0.14	(190)
166	2.59 ± 0.37	(191)
167	1.64 ± 0.15	(191)
465	0.79 ± 0.032	(188)
476	2.61 ± 0.91	(202)
477	3.86 ± 1.03	(202)
479	3.79 ± 1.01	(202)

5.15. Cholinergic Antagonist

Prorocentrolide A (144) blocked both muscle and neuronal nAChRs but with higher affinity on the muscle-type nAChR¹⁶² (affinity constant for muscle-type nAChR was 81.7¹⁶³). Gymnodimine (A) (258) in isolated mouse phrenic hemidiaphragm preparations produced a concentration- and time-dependent block of twitch responses evoked by nerve stimulation without affecting directly elicited muscle twitches, suggesting that it may block the muscle nAChR (EC₅₀ = 10.2 ± 0.7 nM).³¹³ Gymnodimine (A) at 10 nM also blocked, in a voltage-independent manner, homomeric human α7 nAChR expressed in Xenopus oocytes.³¹³ Pinnatoxins bound to the neuronal α7 and muscle-type α1₂βγδ nAChRs.³⁵⁶ The toxins are also bound to the nAChR surrogate, AChBP.³⁵⁶ Pinnatoxin A (293) blocked ACh-evoked currents in oocytes expressing the human α7 nAChR with an IC₅₀ value of 0.107 nM.³⁵⁰ Pinnatoxin G (299) also blocked ACh-evoked currents in oocytes expressing the human α7 nAChR with an IC₅₀ value of 5.06 nM.³⁵⁰ It may be possible to search for skeletal muscular relaxants from phytoplankton toxins.^{162,163,313,350,356}

5.16. Cholinergic Agonists

Anatoxin-a (326) was found to be a potent nicotinic agonist that can produce neuromuscular blockade and death by respiratory arrest.³⁶⁹ Anatoxin-a, with a potency of 31 nM, was identified as an active site-directed inhibitor of acetylcholinesterase. Furthermore, its resistance to oxime reactivation can be attributed to the specific structure of the enzyme adduct it forms.³⁷⁵ The only effective antagonists against a lethal dose of compound anatoxin-a were in vivo pretreatment with physostigmine and high concentrations of 2-PAM.³⁷⁵

5.17. Cannabinoid Receptor Binding Activity

CB₁ is a promising target for a number of diseases, including obesity, neuropathic pain, multiple sclerosis, and post-traumatic stress disorder (PTSD), among others.⁶⁹² Some phytoplankton toxins exhibit moderate CB₁ binding activity,^377,378 some of which may become leading candidates in the search for new drugs. The K_i values of semiplenamides A (337), C (339), and G (343) for CB₁ were 19.5 ± 7.8, 18.7 ± 4.6, and 17.9 ± 5.2 μM, respectively.³⁷⁸ Grenadamide (346) exhibited CB₁ binding activity (K_i = 4.7 μM).³⁷⁷

5.18. Enzyme Inhibitor

Some polypeptides, such as aeruginosins, can inhibit key proteases in several disease processes (Table 4).^{580,581,583,586−588,591,595,596,598} Accordingly, these compounds may become leading compounds in the search for new drugs.

Table 4. Protease Inhibitory Activity of Phytoplankton Toxins.

	IC50
Compound no.	Thrombin	Trypsin	Plasmin	Chymotrypsin	Ref
545	0.3 μg/mL	1.0 μg/mL			(580)
547	7.0 μg/mL	0.6 μg/mL	6.0 μg/mL		(581, 583)
548	10.0 μg/mL	0.6 μg/mL	7.0 μg/mL		(581, 583)
549	3.3 μg/mL	3.9 μg/mL	5.0 μg/mL		(581)
550	3.2 μg/mL	3.0 μg/mL	3.3 μg/mL		(581)
551	0.03 μg/mL	0.4 μg/mL	0.02 μg/mL		(581)
552	0.05 μg/mL	6.6 μg/mL	0.46 μg/mL		(581)
553	0.04 μg/mL	0.2 μg/mL	0.3 μg/mL		(581, 586)
554	0.1 μg/mL	1.1 μg/mL	0.8 μg/mL		(586)
555	9.0 μg/mL	51.0 μg/mL	68.0 μg/mL		(587)
556	1.5 μg/mL	0.07 μg/mL			(588)
557	0.17 μg/mL	0.07 μg/mL			(588)
558		1.9 μM			(591)
559		1.3 μM			(591)
560		19.9 μM			(591)
562		40 μM		>45.5 μM	(595)
563	21.8 nM	112 nM			(596)
534	30 μg/mL	67 μg/mL			(598)
567		45.5 μM			(591)
578		0.09 μM		>45.5 μM	(595)
579		0.62 μM		>45.5 μM	(595)
580		0.09 μM		>45.5 μM	(595)
581		0.65 μM		>45.5 μM	(595)
582		1.12 μM		>45.5 μM	(595)
583		4.27 μM		>45.5 μM	(595)
584		2.01 μM		0.63 μM	(595)
585				0.87 μM	(595)
586				0.22 μM	(595)
587				0.26 μM	(591)

6. Chemical Ecology of HABs

The chemical ecology of HABs is often complex but essential for understanding the environmental effects of HABs. Blooms are subject to macro-predators (fish and invertebrates), micropredators (zooplankton), and competition among co-occurring species. Studies of cyanobacterial blooms concerning the palatability of majusculamides A and B,⁶⁹³ malyngolide B,⁶⁹⁴ laxaphycin A,⁶⁹⁵ ypaoamide,⁶⁹⁶ and pitipeptolide⁶⁹⁷ demonstrated that these metabolites are effective feeding deterrents against a variety of large generalist consumers. Feeding deterrence against micropredators has received less attention. Copepods, common phytoplanktivores, are reported to avoid brevetoxin-rich (359) strains of Karenia brevis under laboratory conditions.⁶⁹⁸ There are ample studies of chemical cues as a form of communication between marine organisms.⁶⁹⁹ The release of copepodamides in the seawater surrounding HABs may serve as chemical cues to warn of the presence of the phytoplanktivores. Copepodamides induce an increase in the production of neurotoxic paralytic shellfish toxins by Alexandrium minutum(700) that can have a severe negative effect on fish.⁷⁰¹ The release of toxic metabolites into the seawater to reduce the fitness of nearest neighbors, allelopathy, was also well documented and may be enhanced by eutrophication.⁷⁰² Water-borne compounds from Karenia brevis were reported to suppress the growth of co-occurring species.⁷⁰³ The copepod Acartia tonsa was also reported to actively avoid dissolved compounds from Karenia brevis in swimming studies.⁷⁰⁴ Among the most interesting controls for HABs is the discovery of the roseobacticides;¹⁹ a novel and highly potent algaecide produced by the α-proteobacteria, Phaeobacter gallaeciencsis and is an effective control of Emiliania huxleyi, a coccolithophorid known to produce large ocean algal blooms.¹⁹ Such experiments mimic the release of compounds in the ocean and begin to address the natural ecology of the bloom, leading to possible biological and natural product controls.

7. Control and Treatment of HABs

Prevention is the preferred management strategy for HABs. Nutrient and greenhouse gas reduction are important strategies. Nevertheless, the implementation of this management strategy can be challenging. There are limited active initiatives specifically dedicated to preventing blooms directly. A burgeoning area of research is dedicated to exploring methods and technologies for controlling or mitigating algal blooms (Figure 6).^705,706 The most ancient and extensively employed physical method for controlling HABs entails using specific clay types during bloom occurrences. The minute and compact clay particles cause the aggregation of HAB cells in the water inducing them to settle until they reach the seabed. The subsequent burial in sediments on the seafloor can also lead to the mortality of the algae cells.⁷⁰⁷ However, several classes of algal toxins have proven stable in sediments for varying lengths of time and resuspension into the water column is possible. Hydrogen peroxide (H₂O₂) and aqueous copper ion (Cu²⁺) have been used as algaecides in some situations.^708,709 As a strong oxidant, H₂O₂ is known for its disinfectant characteristics, as well as its effect on microalgae by inhibiting photosynthetic activity due to damage to the photosystem II.⁷¹⁰ Among HAB species, cyanobacteria such as Microcystis aeruginosa are known to be 10 times more sensitive to H₂O₂e than diatoms and green algae.⁷¹⁰ The breakdown of H₂O₂ has the potential to enhance water quality by oxygenating the water column, thereby promoting the degradation of dissolved organic matter.⁷⁰⁸ Furthermore, it has the capacity to deactivate toxins produced by algal blooms.⁷⁰⁸ Studies have shown that the use of H₂O₂ results in algal commensalism within fish gills.⁷¹¹ This may be a good mitigation strategy for reducing cyanobacteria.⁷¹¹ Additionally, the combination of ultraviolet and visible (UV) light may synergistically enhance the effectiveness of H₂O₂ in aquatic environments.⁷⁰⁸ A dosage lower than 1.6 mg/L of H₂O₂ might even be sufficient for controlling brown tide blooms.^708,712 Micronutrients play a fascinating role in environmental management as even small inputs or changes have the potential to exert significant effects.⁷¹³ For instance, Cu²⁺ has been recommended at concentrations around 100 parts per billion (ppb) as an algal biocide for the management of HABs.⁷¹³ Cu²⁺ at 20 ppb expressed a tremendous stimulatory effect, suggesting that HAB management with Cu²⁺ may not always be advisible.⁷¹³ The use of parasites to infect the dinoflagellates that cause HABs may be an alternative approach to HAB management.⁷¹⁴ However, this approach has had negative, long-term and unintended environmental impacts.⁷¹⁵

8. Conclusion

Phytoplankton, often hailed as “biological carbon pumps,” play an indispensable role in the carbon cycle of the ecosystem due to their rapid growth rates, photosynthetic efficiency, and critical history in the creation of present-day petroleum reserves. Their capacity for carbon sequestration is illustrated by their role in the formation of Earth’s fossil petroleum reserves. This may highlight their potential as a sustainable tool for the sequestration of CO₂ from the environment. Exploring technologies that culture algae for platform chemical production presents a promising avenue for reducing our reliance on fossil fuels as well as mitigating climate change. However, the excessive growth of toxic algae poses serious ecological and economic threats, particularly in the form of HABs. This presents a significant challenge for the fishery and seafood industries. The production of toxic metabolites by HABs can have devastating impacts on ecosystems, food supplies, human health, and regional economies-underscoring the need for effective forecasting, mitigation, and management strategies. The unique structural metabolite classes that phytoplankton produce with potent biological activities and novel mechanisms of action offer unprecedented opportunities for drug discovery. These compounds have the unique claim of being some of the most complex molecules ever characterized and serve as a valuable resource for the development of novel therapeutics. The challenges associated with the structural elucidation of phytoplankton metabolites have led to advancements in computational tools, such as DP4+ calculations, and JBCA for facilitating NMR assignments. These tools, alongside other metabolomics and chemical informatics approaches using mass spectrometry, are crucial for exploring the vast diversity of phytoplankton chemistry.

The dual nature of phytoplankton as both a boon and a bane necessitates a balanced approach to harness their benefits while mitigating exposure to toxins. Significantly more research is required to develop methods that control HABs effectively without compromising the substantial advantages offered by microalgae in carbon management strategies. As we develop sustainable practices it is imperative to continue exploring the vast potential of phytoplankton as a resource to sequester carbon, replace petroleum products, and provide innovative and complex metabolites as therapeutics. This all must be done while protecting the environment and our food supplies. The crucial role of phytoplankton in managing the global carbon cycle and their potential to contribute to novel therapeutic discoveries cannot be overstated. Future efforts should focus on innovative solutions that leverage the benefits of phytoplankton while ensuring the preservation and health of our ecosystems.

Biographies

Xiaoying Liu is currently a graduate student in the Institute of Chinese Medical Sciences at Macau University at Macau University. She received her Bachelor degree at Jinan University in 2020 and Master degree at Lanzhou University. Her current research focuses on natural product chemistry.

Zhiwei Bian is currently a graduate student in the Department of Pharmacy at Lanzhou University. Her current research focuses on natural product chemistry.

Shian Hu is currently a graduate student in the Department of Pharmacy at Lanzhou University. His current research focuses on natural product chemistry.

Cody F. Dickinson obtained his Ph.D. in Organic Chemistry from the University of Hawaii under the supervision of Prof. Marcus Tius. He is currently a Staff Scientist in Prof. Mark Hamann’s lab at the Medical University of South Carolina. His current research interests are focused on the synthesis of complex natural products.

Menny M. Benjamin obtained his M.S. degree in Biomedical Sciences from the Medical University of South Carolina under the mentorship of Dr. Mark T. Hamann. He is currently conducting natural product research for the isolation of antitumor, antibacterial, and antiviral secondary metabolites from marine and terrestrial specimens in Prof. Hamann’s laboratory.

Jia Jia is an associate professor at Shanghai University. He completed his Ph.D. at Clemson University. His research interests focus on biomedical engineering and biochemistry.

Yintai Tian is currently a graduate student in the Department of Pharmacy at Lanzhou University. He received his Bachelor’s degree at China Pharmaceutical University in 2020. His current research focuses on the natural product chemistry.

Allen Place is a faculty member with the Institute of Marine and Environmental Technology and is an expert in the culture of phytoplankton and the generation of their associated toxic molecules.

George S. Hanna obtained his Ph.D. in Biomedical Sciences from the Medical University of South Carolina under the mentoring of Dr. Mark Hamann. In 2023 he joined the Medical University of South Carolina’s College of Medicine and operates a lab in the Holling’s Marine Lab focused on characterizing the biological activities of natural products for therapeutic development.

Hendrik Luesch is a Professor of Medicinal Chemistry at the University of Florida. He leads a multidisciplinary marine natural products program with chemistry and genomics-based discovery platforms.

Peter Croot is a marine biogeochemist with the University of Galway Ireland. His lab studies the distribution of trace elements and unique chemical species in the Oceans.

Maggie M. Reddy is part of the School of Biological and Chemical Sciences at the Ryan Insitute at the University of Galway Ireland. Her lab studies metabolomic and multiomics approaches to characterize algal metabolism.

Olivier P. Thomas is a Professor of marine biodiscovery at the National University of Ireland’s Ryan Institute. His lab focuses on the characterization of unique marine natural products including HAB toxins from phytoplankton.

Gary Hardiman is a Professor with the Faculty of Medicine, Health and Life Sciences at Queen’s University Ireland and his lab studies ocean systems biology.

Melany P. Puglisi is a Professor of Pharmaceutical Sciences at Chicago State University and her lab studies the chemical ecology of HAB toxins.

Ming Yang is an Assistant Professor with the Department of Chemical and Biomolecular Engineering at Clemson University. His lab studies reaction engineering for energy and environmental applications.

Zhi Zhong received her MS and MD degree from Sun Yat-Sen University in Medical Sciences and PhD and Postdoctoral training in nutrition, hepatology and toxicology at UNC Chapel Hill. Her lab studies liver disease.

John J. Lemasters served as a faculty member at UT Southwestern, UNC Chapel Hill before joining MUSC. His lab studies liver disease and leads the microscopy core at MUSC.

Jeffrey E. Korte is a faculty member of public health with MUSC and his lab studies infectious diseases and HPV related cancer.

Amanda L. Waters serves as a faculty member of the University of Central Oklahoma and completed graduate and postdoctoral traning in the chemistry of HAB toxins and other natural products.

Carl E. Heltzel is an Associate Professor with MUSC’s Hollings Cancer Center and serves as the Science Director and Editor of Bioprobe, a quarterly newsletter.

R. Thomas Williamson is the Yousry Sayed Distinguished Professor of Pharmaceutical Sciences at UNC Willmington. His lab is a leader in the development and application of structure assignment tools for complex HAB toxins.

Wendy K. Strangman, after completing her Ph.D. at the Scripps Institution of Oceanography in Marine Natural Products Chemistry and postdoctoral research at the University of British Columbia, Wendy joined UNCW as a Research Associate Professor at the Center for Marine Science. Research in her group focuses on discovery of new biologically active natural products for therapeutic development as well as detection and characterization of new toxins produced by harmful algal blooms.

Fred Valeriote is an in vitro and in vivo pharmacologist at the Ford Cancer Center in Detroit and his lab studies phytochemicals that prevent or promote the development of cancer.

Marcus A. Tius is a Professor of Chemistry at the University of Hawaii and Member of the Hawaii Cancer Center. His lab focuses on the synthesis of complex alkaloids.

Jack DiTullio completed a PhD in Oceanography at the University of Hawaii at Manoa. He is currently a professor of Oceanography at the College of Charleston and his lab studies the impact of phytoplankton on the oceans.

Daneel Ferreira served as Professor of Chemistry with the University of the Free State Bloemfoutein, South Africa and is currently Professor Emeritus with the University of Mississippi School of Pharmacy and National Center for Natural Products Research.

Alexander Alekseyenko is a Faculty Member in Public Health at MUSC and his lab studies environmental informatics.

Shengpeng Wang obtained his Ph.D. in Traditionally Chinese Medicine from the University of Macau and currently he is an assistant professor at University of Macau. His research interests focus on Traditional Chinese Medicine and nanomedicine.

Mark T. Hamann obtained his Ph.D. in Organic Chemistry from the University of Hawaii and joined the faculty at the University of Mississippi School of Pharmacy and Chemistry & Biochemistry Department. In 2016 he joined the Medical University of South Carolina’s College of Pharmacy, College of Medicine and Hollings Cancer Center.

Xiaojuan Wang completed her Ph.D. under the supervision of Dr. Mark Hamann at Medical University of South Carolina. After working at Princeton University as a postdoc in 2019, she joined Lanzhou University in 2020. Her current research interests focus on natural product chemistry and its implications for human health.

Author Contributions

^# X.L., Z.B., S.H., C.F.D., and M.M.B. contributed equally to this work. CRediT: Shian Hu data curation; Yintai Tian data curation; Carl Heltzel writing - review & editing; Menny M. Benjamin image generation, review, & editing; Mark T. Hamann and Xiajuan Wang conceptualization, data curation, formal analysis, funding acquisition, investigation, methodology, project administration, resources, supervision, validation, visualization, writing - original draft, writing - review & editing; J.D. data curation, writing - review & editing; J.J., Y.T., A.P., G.S.H., H.L., P.C., M.M.R., O.P.T., G.H., M.P.P., M.Y., Z.Z., J.J.L., J.E.K., A.L.W., C.E.H., R.T.W., W.K.S., F.V., M.A.T., D.F., A.A., and S.W. writing - review & editing.

The authors declare no competing financial interest.