3 research outputs found
Epimers of Azaspiracids: Isolation, Structural Elucidation, Relative LC-MS Response, and <i>in Vitro</i> Toxicity of 37-<i>epi</i>-Azaspiracid‑1
Since
azaspiracid-1 (AZA1) was identified in 1998, the number of
AZA analogues has increased to over 30. The development of an LC-MS
method using a neutral mobile phase led to the discovery of isomers
of AZA1, AZA2, and AZA3, present at ∼2–16% of the parent
analogues in phytoplankton and shellfish samples. Under acidic mobile
phase conditions, isomers and their parents are not separated. Stability
studies showed that these isomers were spontaneous epimerization products
whose formation is accelerated with the application of heat. The AZA1
isomer was isolated from contaminated shellfish and identified as
37-<i>epi</i>-AZA1 by nuclear magnetic resonance (NMR) spectroscopy
and chemical analyses. Similar analysis indicated that the isomers
of AZA2 and AZA3 corresponded to 37-<i>epi</i>-AZA2 and
37-<i>epi</i>-AZA3, respectively. The 37-epimers were found
to exist in equilibrium with the parent compounds in solution. 37-<i>epi</i>-AZA1 was quantitated by NMR, and relative molar response
studies were performed to determine the potential differences in LC-MS
response of AZA1 and 37-<i>epi</i>-AZA1. Toxicological effects
were determined using Jurkat T lymphocyte cells as an <i>in vitro</i> cell model. Cytotoxicity experiments employing a metabolically based
dye (i.e., MTS) indicated that 37-<i>epi</i>-AZA1 elicited
a lethal response that was both concentration- and time-dependent,
with EC<sub>50</sub> values in the subnanomolar range. On the basis
of EC<sub>50</sub> comparisons, 37-<i>epi</i>-AZA1 was 5.1-fold
more potent than AZA1. This data suggests that the presence of these
epimers in seafood products should be considered in the analysis of
AZAs for regulatory purposes
Structure Elucidation, Relative LC–MS Response and In Vitro Toxicity of Azaspiracids <b>7</b>–<b>10</b> Isolated from Mussels (Mytilus edulis)
Azaspiracids
(AZAs) are marine biotoxins produced by dinoflagellates that can accumulate
in shellfish, which if consumed can lead to poisoning events. AZA7–10, <b>7</b>–<b>10</b>, were isolated from shellfish and
their structures, previously proposed on the basis of only LC–MS/MS
data, were confirmed by NMR spectroscopy. Purified AZA4–6, <b>4</b>–<b>6</b>, and <b>7</b>–<b>10</b> were accurately quantitated by qNMR and used to assay cytotoxicity
with Jurkat T lymphocyte cells for the first time. LC–MSÂ(MS)
molar response studies performed using isocratic and gradient elution
in both selected ion monitoring and selected reaction monitoring modes
showed that responses for the analogues ranged from 0.3 to 1.2 relative
to AZA1, <b>1</b>. All AZA analogues tested were cytotoxic to
Jurkat T lymphocyte cells in a time- and concentration-dependent manner;
however, there were distinct differences in their EC<sub>50</sub> values,
with the potencies for each analogue being: AZA6 > AZA8 > AZA1
> AZA4 ≈ AZA9 > AZA5 ≈ AZA10. This data contributes
to the understanding of the structure–activity relationships
of AZAs
Isolation, Structure Elucidation, Relative LC-MS Response, and in Vitro Toxicity of Azaspiracids from the Dinoflagellate <i>Azadinium spinosum</i>
We identified three new azaspiracids
(AZAs) with molecular weights
of 715, 815, and 829 (AZA33 (<b>3</b>), AZA34 (<b>4</b>), and AZA35, respectively) in mussels, seawater, and <i>Azadinium
spinosum</i> culture. Approximately 700 μg of <b>3</b> and 250 μg of <b>4</b> were isolated from a bulk culture
of <i>A. spinosum</i>, and their structures determined by
MS and NMR spectroscopy. These compounds differ significantly at the
carboxyl end of the molecule from known AZA analogues and therefore
provide valuable information on structure–activity relationships.
Initial toxicological assessment was performed using an in vitro model
system based on Jurkat T lymphocyte cytotoxicity, and the potencies
of <b>3</b> and <b>4</b> were found to be 0.22- and 5.5-fold
that of AZA1 (<b>1</b>), respectively. Thus, major changes in
the carboxyl end of <b>1</b> resulted in significant changes
in toxicity. In mussel extracts, <b>3</b> was detected at low
levels, whereas <b>4</b> and AZA35 were detected only at extremely
low levels or not at all. The structures of <b>3</b> and <b>4</b> are consistent with AZAs being biosynthetically assembled
from the amino end