Description: The harmful algal bloom(HAB)-forming species Alexandrium ostenfeldii produces paralytic shellfish poisoning (PSP) toxins and the lesser known spiroimine shellfish poisoning (SSP) toxins. These toxins primarily belong to macrocyclic imine class, with cyclic imine moiety and macrocyclic nature as their distinct characteristics. By far, A. ostenfeldii is the only species identified to produce two types of cyclic imines—gymnodimines and spirolides. Studies on these toxins produced by different A. ostenfeldii strains revealed high structural diversity. The presence of highly varied structural types of cyclic imines that requires elaborate synthesis pathways implies its underlying function for the producing organism. However, known roles and mode of action (MOA) of cyclic imines are rather limited. That is, other than its “fasting-acting” toxicity and antagonistic activity against of nicotinic acetylcholine receptors, no other MOA of cyclic imines have been identified. Considering the increasing occurrence of HABs and the socio-economic impacts of toxins associated in A. ostenfeldii, it is crucial to elucidate not only the structural diversity of cyclic imines but also their respective modes of action and biological significance of their production. As such, this study aimed to isolate, characterize, and screen the bioactivity of the two types of cyclic imines (i.e., gymnodimines and spirolides) produced by A. ostenfeldii. Two strains of A. ostenfeldii (OKNL 48 and X-LF-19-F10) were mass cultivated to directly obtain purified samples of structurally known gymnodimines (i.e., gymnodimine A (GYM A)) and spirolides (i.e., 13-desmethyl spirolide C (SPX 1)). A total of 345 µg of GYM A and 559 µg of SPX 1 were purified (〉95% purity) from the large-scale microalgal culture. Three other A. ostenfeldii strains (AON 24, NX-56-10 and MX-S-B11) were identified to produce both structurally unknown spirolides. Isolation of the pure novel spirolides were not possible in quantities necessary for NMR structure elucidation work. Thus, mass-spectrometric (MS)-based techniques was sought as an alternative approach for the structural characterization of unknown compounds. Using the technique, this study revealed the presence of nine novel spirolides, eight of which have either a 5:5:6 (C-type spirolides) or 5:6:6 (G-type spirolides) triketal ring configuration. Another novel spirolide putatively belongs to a new spirolide subclass, which has a 6:5:6 triketal ring configuration that is only observed in pinnatoxins, another class of cyclic imines. The initial structural insights by mass spectral techniques demonstrate that structural variability of spirolides is not only limited to the presence or absence of certain functional groups (as observed in other eight novel spirolides) but also the triketal ring system. Moreover, the proposed 6:5:6 triketal ring system in one of the novel spirolides suggests that similar biosynthetic steps are involved in the synthesis of spirolides and pinnatoxins. To gain a better understanding on the potential role of cyclic imines, purified GYM A and SPX 1 were subjected into cell line-based bioassays to identify other MOA of the toxins. Intracellular calcium levels ([Ca]i) measured in rat pheochromocytoma (PC12) cells suggested that both toxins elicited differential effect on acetylcholine receptor (AChRs) subtypes. GYM A and SPX 1 activates nicotinic AChRs (nAChRs) while only GYM A activate muscarinic AChRs (mAChRs). The activation of AChRs and the subsequent influx of calcium ions into the cell illustrates the putative capacity of the toxins to mimic acetylcholine. These observations were possible as a result of determining the response of GYM A or SPX 1 on [Ca]i signaling in PC12 cells. Moreover, preliminary investigations on the effects of GYM A and SPX 1 to AREc32 and Nf-κB-bla THP-1 cell lines indicated that toxins potentially induce adaptive stress response pathways. SPX 1 likely activates both oxidative stress response and inflammation response pathways. GYM A, on the other hand, possibly initiates inflammation response pathways but lacks the capacity to induce oxidative stress response pathway. The ability of the toxins to potentially activate adaptive response pathways indicated that GYM A or SPX 1 might initiate cellular processes that will restore homeostasis suggesting possible medicinal applications of the toxins. Studies on the response of GYM A and SPX 1 to [Ca]i and adaptive stress response pathways provided more understanding on the potential MOA of the toxins. Further knowledge on structural diversity and MOA of cyclic imines could hopefully direct us to determine structure-activity relationship and, to some extent, purpose of the toxins to the producing organism.

Description: Spirolides belong to a group of marine phycotoxins produced by the marine planktonic dinophyte Alexandrium ostenfeldii. Composed of an imine moiety and a spiroketal ring system within a macrocylcle, spirolides are highly diverse with toxin types that vary among different strains. This study aims to characterize the spirolides from clonal A. ostenfeldii strains collected from the Netherlands, Greenland and Norway by mass spectral techniques. The structural characterization of unknown spirolides as inferred from high-resolution mass spectrometry (HR-MS) and collision induced dissociation (CID) spectra revealed the presence of nine novel spirolides that have the pseudo-molecular ions m/z 670 (1), m/z 666 (2), m/z 696 (3), m/z 678 (4), m/z 694 (5), m/z 708 (6), m/z 720 (7), m/z 722 (8) and m/z 738 (9). Of the nine new spirolides proposed in this study, compound 1 was suggested to have a truncated side chain in lieu of the commonly observed butenolide ring in spirolides. Moreover, there is indication that compound 5 might belong to new spirolide subclasses with a trispiroketal ring configuration having a 6:5:6 trispiroketal ring system. On the other hand, the other compounds were proposed as C- and G-type SPX, respectively. Compound 7 is proposed as the first G-type SPX with a 10-hydroxylation as usually observed in C-type SPX. This mass spectrometry-based study thus demonstrates that structural variability of spirolides is larger than previously known and does not only include the presence or absence of certain functional groups but also involves the triketal ring system.

Description: The harmful algal bloom(HAB)-forming species Alexandrium ostenfeldii produces paralytic shellfish poisoning (PSP) toxins and the lesser known spiroimine shellfish poisoning (SSP) toxins. These toxins primarily belong to macrocyclic imine class, with cyclic imine moiety and macrocyclic nature as their distinct characteristics. By far, A. ostenfeldii is the only species identified to produce two types of cyclic imines—gymnodimines and spirolides. Studies on these toxins produced by different A. ostenfeldii strains revealed high structural diversity. The presence of highly varied structural types of cyclic imines that requires elaborate synthesis pathways implies its underlying function for the producing organism. However, known roles and mode of action (MOA) of cyclic imines are rather limited. That is, other than its “fasting-acting” toxicity and antagonistic activity against of nicotinic acetylcholine receptors, no other MOA of cyclic imines have been identified. Considering the increasing occurrence of HABs and the socio-economic impacts of toxins associated in A. ostenfeldii, it is crucial to elucidate not only the structural diversity of cyclic imines but also their respective modes of action and biological significance of their production. As such, this study aimed to isolate, characterize, and screen the bioactivity of the two types of cyclic imines (i.e., gymnodimines and spirolides) produced by A. ostenfeldii. Two strains of A. ostenfeldii (OKNL 48 and X-LF-19-F10) were mass cultivated to directly obtain purified samples of structurally known gymnodimines (i.e., gymnodimine A (GYM A)) and spirolides (i.e., 13-desmethyl spirolide C (SPX 1)). A total of 345 μg of GYM A and 559 μg of SPX 1 were purified (〉95% purity) from the large-scale microalgal culture. Three other A. ostenfeldii strains (AON 24, NX-56-10 and MX-S-B11) were identified to produce both structurally unknown spirolides. Isolation of the pure novel spirolides were not possible in quantities necessary for NMR structure elucidation work. Thus, mass-spectrometric (MS)-based techniques was sought as an alternative approach for the structural characterization of unknown compounds. Using the technique, this study revealed the presence of nine novel spirolides, eight of which have either a 5:5:6 (C-type spirolides) or 5:6:6 (G-type spirolides) triketal ring configuration. Another novel spirolide putatively belongs to a new spirolide subclass, which has a 6:5:6 triketal ring configuration that is only observed in pinnatoxins, another class of cyclic imines. The initial structural insights by mass spectral techniques demonstrate that structural variability of spirolides is not only limited to the presence or absence of certain functional groups (as observed in other eight novel spirolides) but also the triketal ring system. Moreover, the proposed 6:5:6 triketal ring system in one of the novel spirolides suggests that similar biosynthetic steps are involved in the synthesis of spirolides and pinnatoxins. To gain a better understanding on the potential role of cyclic imines, purified GYM A and SPX 1 were subjected into cell line-based bioassays to identify other MOA of viii the toxins. Intracellular calcium levels ([Ca]i) measured in rat pheochromocytoma (PC12) cells suggested that both toxins elicited differential effect on acetylcholine receptor (AChRs) subtypes. GYM A and SPX 1 activates nicotinic AChRs (nAChRs) while only GYM A activate muscarinic AChRs (mAChRs). The activation of AChRs and the subsequent influx of calcium ions into the cell illustrates the putative capacity of the toxins to mimic acetylcholine. These observations were possible as a result of determining the response of GYM A or SPX 1 on [Ca]i signaling in PC12 cells. Moreover, preliminary investigations on the effects of GYM A and SPX 1 to AREc32 and Nf-κB-bla THP-1 cell lines indicated that toxins potentially induce adaptive stress response pathways. SPX 1 likely activates both oxidative stress response and inflammation response pathways. GYM A, on the other hand, possibly initiates inflammation response pathways but lacks the capacity to induce oxidative stress response pathway. The ability of the toxins to potentially activate adaptive response pathways indicated that GYM A or SPX 1 might initiate cellular processes that will restore homeostasis suggesting possible medicinal applications of the toxins. Studies on the response of GYM A and SPX 1 to [Ca]i and adaptive stress response pathways provided more understanding on the potential MOA of the toxins. Further knowledge on structural diversity and MOA of cyclic imines could hopefully direct us to determine structure-activity relationship and, to some extent, purpose of the toxins to the producing organism.