Structural elucidation and quantification of novel toxins in marine microalgae by NMR- and molecular modelling-based techniques

Marine biotoxins produced by dinoflagellates exhibit a remarkable structural diversity. During the course of this thesis, I present the elucidation of six novel compounds in total, two for each of the toxin groups of azaspiracids, gymnodimines, and spirolides. The LC-MS based quantification, as used in shellfish surveillance, of the novel azaspiracids was compared with NMR based quantification. Further, four cyclic imine toxins had their full relative stereochemistry elucidated. The simulated chemical shifts revealed a systematic effect between the chirality at C 4 and neighboring nuclei. Based on this systematic effect, reasonable doubts arise towards determination of R configuration at C-4 in 13,19-didesmethyl SPX C. The configuration of C-4 was assigned with simulated and measured NMR spectra, suggesting that all tested GYMs and SPXs have the S configuration at that position. This assignment supports the hypothesis of a common biosynthetic pathway for both compound groups

In Silico Modeling of Spirolides and Gymnodimines: Determination of S Configuration at Butenolide Ring Carbon C-4

Only few naturally occurring cyclic imines have been fully structurally elucidated or synthesized to date. The configuration at the C-4 carbon plays a pivotal role in the neurotoxicity of many of these metabolites, for example, gymnodomines (GYMs) and spirolides (SPXs). However, the stereochemistry at this position is not accessible by nuclear Overhauser effect—nuclear magnetic resonance spectroscopy (NOE-NMR) due to unconstrained rotation of the single carbon bond between C-4 and C-5. Consequently, the relative configuration of GYMs and SPXs at C-4 and its role in protein binding remains elusive. Here, we determined the stereochemical configuration at carbon C-4 in the butenolide ring of spirolide- and gymnodimine-phycotoxins by comparison of measured 13C NMR shifts with values obtained in silico using force field, semiempirical and density functional theory methods. This comparison demonstrated that modeled data support S configuration at C-4 for all studied SPXs and GYMs, suggesting a biosynthetically conserved relative configuration at carbon C-4 among these toxins

Selective purification of catecholate, hydroxamate and α-hydroxycarboxylate siderophores with titanium dioxide affinity chromatography

Siderophores, high affinity iron chelators, play a key role in the uptake of iron by microorganisms and regulate many biological functions. Siderophores are categorized by their chelating group, e.g., catecholates, hydroxamates, α-hydroxycarboxylates. Natural concentrations of siderophores are often either too low or sample matrices are too complex for direct analysis by, e.g., liquid chromatography – mass spectrometry. Therefore, both concentration and purification are prerequisite for reliable analyses. However, a chromatographic technique that is selective for all siderophore classes and affords high levels of purification is lacking. We developed a titanium dioxide affinity chromatography (TDAC) solid-phase extraction (SPE) that affords the selective purification of these siderophore classes from complex sample matrices with recoveries up to 82%. The one-step purification removed most non-ligand sample ‘contaminants’, therefore, affording the straightforward identification of siderophore peaks in base peak chromatograms. As a proof of concept, the bioinformatic processing, dereplication of known features and selection of significant features in the TDAC eluates afforded a fast identification of six novel siderophores (woodybactines) from bacterial supernatants. We propose TDAC SPE as a fast and cost-effective methodology to screen for known or discover novel siderophores in natural samples in combination with untargeted bioinformatic processing by, e.g., XCMS. The method is scalable and yielded large amounts of highly purified siderophores from bacterial culture supernatants, providing an effective quantitative sample clean-up for, e.g., NMR structure elucidation

Structure and toxicity of AZA-59, an azaspiracid shellfish poisoning toxin produced by Azadinium poporum (Dinophyceae)

To date, the putative shellfish toxin azaspiracid 59 (AZA-59) produced by Azadinium poporum (Dinophyceae) has been the only AZA found in isolates from the Pacific Northwest coast of the USA (Northeast Pacific Ocean). Anecdotal reports of sporadic diarrhetic shellfish poisoning-like illness, with the absence of DSP toxin or Vibrio contamination, led to efforts to look for other potential toxins, such as AZAs, in water and shellfish from the region. A. poporum was found in Puget Sound and the outer coast of Washington State, USA, and a novel AZA (putative AZA-59) was detected in low quantities in SPATT resins and shellfish. Here, an A. poporum strain from Puget Sound was mass-cultured and AZA-59 was subsequently purified and structurally characterized. In vitro cytotoxicity of AZA-59 towards Jurkat T lymphocytes and acute intraperitoneal toxicity in mice in comparison to AZA-1 allowed the derivation of a provisional toxicity equivalency factor of 0.8 for AZA-59. Quantification of AZA-59 using ELISA and LC-MS/MS yielded reasonable quantitative results when AZA-1 was used as an external reference standard. This study assesses the toxic potency of AZA-59 and will inform guidelines for its potential monitoring in case of increasing toxin levels in edible shellfish

Strukturaufklärung und Quantifizierung von neuen Toxinen in marinen Mikroalgen durch NMR und Molekular Modelling basierten Techniken

Marine biotoxins produced by dinoflagellates exhibit a remarkable structural diversity. During the course of this thesis, I present the elucidation of six novel compounds in total, two for each of the toxin groups of azaspiracids, gymnodimines, and spirolides. The LC-MS based quantification, as used in shellfish surveillance, of the novel azaspiracids was compared with NMR based quantification. Further, four cyclic imine toxins had their full relative stereochemistry elucidated. The simulated chemical shifts revealed a systematic effect between the chirality at C 4 and neighboring nuclei. Based on this systematic effect, reasonable doubts arise towards determination of R configuration at C-4 in 13,19-didesmethyl SPX C. The configuration of C-4 was assigned with simulated and measured NMR spectra, suggesting that all tested GYMs and SPXs have the S configuration at that position. This assignment supports the hypothesis of a common biosynthetic pathway for both compound groups