Di Matteo, Viviana (2025) An innovative platform for the discovery of new marine bioactive natural substances. [Tesi di dottorato]

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Abstract

Research on natural products continues to link molecular discoveries with ecological and biomedical knowledge, but progress often stalls at the crossroads between complex mixtures and structural certainties. This thesis proposes and demonstrates an integrated pathway that flows smoothly from genes to networks, to spectra, and, when necessary, to landscapes. High-resolution LC-MS/MS, multidimensional NMR, and targeted derivatization strategies provide the analytical framework; feature-based molecular networking focuses attention on coherent families rather than isolated peaks; and genome mining provides a biosynthetic rationale that governs structural assignments and selects what should be isolated. Throughout the work, this combination allows for early dereplication, more precise targeted isolation, and more robust justification of stereochemical assignments, while opening up pathways to link molecules to function in living systems. The scientific results of this thesis are described in the chapters 5, 7 and 8. The first section focuses on a family of lipophilic cyclohexapeptides produced by a thermophilic bacterium. Starting from a classic antibacterial assay, the study reexamines thermoactinamide A and reveals a broader constellation of congeners that were previously obscured by low abundance and spectral overlap. Genome sequencing reveals a greater than expected capacity for non-ribosomal peptide synthesis, while domain predictions help reconcile MS/MS fragmentation patterns with plausible sequences. Molecular networking highlights weak but chemically consistent signals; taken together, these tools expand the thermoactinamide family and highlight flexible and potentially iterative behaviour within specific NRPS modules. The story then moves on to synthesis, not simply as a source of material, but as proof of truth. Under widely used PyBOP-mediated macrocyclization conditions, the synthesized thermoactinamide does not match the natural compound. NMR comparison, enhanced Marfey analysis, and LC-HRMS demonstrate that the macrocyclization reaction reverses the α stereogenic centre of the activated residue during ring closure, thus questioning the paradigm of chemical synthesis as the final proof of structure. This discovery accounts for the discrepancies present in the literature and has broader implications: if macrocyclization may alter the stereochemistry of the target, then an entire structure-activity campaign risks being built on epimeric surrogates. The second section presents the discovery and determination of the stereochemical structure of cyanochelin B, a photolabile siderophore produced by a filamentous cyanobacterium. High-resolution mass spectrometry and comprehensive 1D/2D NMR establish the planar structure. Genome mining identify a hybrid NRPS-PKS cluster whose predicted adenylation specificities matched the observed structure and the unusual presence of β-hydroxyaspartate. The absolute configuration of the molecule is elucidated using Marfey derivatization and NMR-based Murata method, complemented with bioinformatic analysis. Functionally, the Fe(III) complex of cyanochelin B undergoes rapid photolysis under UV-A light, fragmenting into a small set of defined products while simultaneously photoreducing iron from Fe(III) to Fe(II). In a membrane co-culture, the siderophore privatizes iron in the dark but loses its exclusivity under illumination, allowing a non-producing competitor to capture iron and grow more rapidly. This light-dependent mechanism links daytime irradiation to the microbial iron economy: the same molecule that imposes a monopoly at sunset becomes a channel for sharing at midday. The distribution of the biosynthetic gene cluster within related cyanobacteria, sometimes on mobile elements, suggests that this strategy is both transferable and responsive to environments where iron limitation intersects with strong light fluctuations. By uncovering a mechanistic link between photochemistry and nutrient flux, the study calls for a reinterpretation of laboratory competition experiments that ignore light regimes and proposes ecological investigations along depth and clarity gradients to map where siderophores act. The third section extends the workflow from the lab benches to a real-world setting during an episode in which a small volcanic lake took on an unexpected red hue. A rapid detection strategy combines retrospective satellite screening with targeted UAV surveys to locate and quantify surface heterogeneity. Field teams then collect samples for microscopy, amplicon sequencing, and untargeted metabolomics. Feature-based molecular networking quickly organizes the resulting spectra, revealing that the bloom's metabolome is dominated by anabaenopeptins, including several variants not previously reported at the site. This chemical fingerprint is not confined to the lake: hydrological connectivity carries the signal downstream to a coastal area with shellfish aquaculture, transforming an optical anomaly into a food safety issue. The case demonstrates that multiscale detection combined with rapid chemotyping can reduce the time between detection and assessment. Overall, the thesis proposes a practice rather than a single technique. It demonstrates that the search for novelty should start with families and biosynthetic expectations; that synthesis should be used not only to create molecules, but also to test our structural claims; and that environmental questions are best answered when optics and omics engage in dialogue with each other. From a methodological point of view, this approach reduces friction in research and protects against reassuring but erroneous structural narratives. The broader message is that the shortest line from nature to insight is triangular, connecting genomes, networks, and spectra.

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