The rediscovery of known drug classes represents a major challenge in natural products drug discovery. Compound rediscovery inhibits the ability of researchers to explore novel natural products and wa...

Bromotyrosine-containing natural products have been isolated from marine sponges. While studying the uptake of engineered bacterial strains by sponges, we observed the production of fistularin-3 in the sponge. To explore these results, we employed multimodal imaging to colocalize metabolites with fluorescent in situ hybridization (FISH) probes. Using mass spectrometry imaging (MSI), we measured fistularin-3 and mapped this against the FISH probes to locate the bacteria. A recent study reported that P. brasiliensis produced fistularin-3 in pure culture, but these data have not yet been reproduced. We employed MSI and FISH to colocalize the fistularin-3 spatial distribution with P. brasiliensis in sponge tissue.

Asian and American Aconitum species are phylogenetically close, but only certain Asian species have been well-studied for their medicinal properties. This study aims to discover bioactive compounds in...

Metal chelating small molecules (metallophores) play significant roles in microbial interactions and bacterial survival; however, current methods to identify metallophores are limited by low sensitivity, a lack of metal selectivity, and/or complicated data analysis. To overcome these limitations, we developed a novel approach for detecting metallophores in natural product extracts using ion mobility-coupled mass spectrometry (IM-MS). As a proof of concept, marine bacterial extracts containing known metallophores were analyzed by IM-MS with and without added metals, and the data were compared between conditions to identify metal-binding metabolites. Ions with changes in both mass and mobility were specific to metallophores, enabling their identification within these complex extracts. Additionally, we compared the use of direct infusion (DI) and liquid chromatography (LC) separation with IM-MS. For most samples, DI outperformed LC by minimizing the time required for data collection and simplifying analysis. However, for some samples, LC improved the detection of metallophores likely by reducing ion suppression. IM-MS was then used to identify 10 metallophores in an extract from a marine Micromonospora sp. Overall, incorporating IM-MS facilitated the rapid detection of metal-binding natural products in complex bacterial extracts through the comparison of mass and mobility data in the presence and absence of metals.

An advanced metabolomic workflow featuring expert knowledge-tailored mass spectral queries and multi-informative molecular networking was applied to French DNA-barcoded collections of Amanita species belonging to the section Phalloideae. The study aimed to clarify the toxin content of these mushrooms, focusing on chemical differences between A. virosa and A. amerivirosa which lacked available state-of-the-art analytical data. We validated the inability of A. amerivirosa to produce α-amanitin but revealed that it synthesizes amaninamide, quantified herein for the first time. Careful evaluation of the MS/MS fragmentation patterns associated with ama-, phallo-, and virotoxins led to interoperable MassQL queries (viz., compatible with MS/MS spectra from both QTOF and Orbitrap) for identifying these toxins in MS/MS data sets, with varying degrees of structural accuracy. The ability of these queries to convey structural information was highlighted by the MS/MS prediction of six new toxins, with structural proposals validated after isolation and structure elucidation by NMR. Among the reported phallotoxins, ameriphalloidin (1) displayed an unprecedented viroidin-like amino acid sequence, supporting the often-inferred biosynthetic relationship between these toxin classes. The discovery of new phallo- and virotoxins is significant, as these are the first Amanita toxins to be reported since the pioneering work of Wieland and Faulstich between 1930 and 1980.

This study established LC-MS-based metabolomics integrated with multivariate statistical analysis (UMMMS) to identify differential metabolites in Actinoalloteichus cyanogriseus under solid versus liquid culture conditions. Using this approach, 48 molecules exhibiting significant abundance differences between solid and liquid media were rapidly screened out. Among these, 8 were preliminarily identified as peptides potentially containing the unique β-Me-Leu residue by targeted MS/MS analysis. Guided by these screening results, 5 previously undiscovered peptides, designated as cyanogristatins A–E (1–5), were isolated. Their structures were elucidated using 1D/2D NMR spectroscopy, HR-MS/MS, and the advanced Marfey’s method. Furthermore, 5 effectively reduced the number of migrating inflammatory cell aggregates in a zebrafish model of inflammatory bowel disease at a concentration of 20 μM. In subsequent experiments using an in vitro organoid model, 5 did not exert direct epithelial protective effects; however, ELISA further revealed that it decreased the level of production of TNF-α in intestinal macrophages. This finding suggests that the anti-inflammatory effect of 5 is most likely mediated by the regulation of the cytokine TNF-α. In summary, this study demonstrates the potential of mass spectrometry technology for the comprehensive analysis of natural products, facilitating the rapid discovery of novel compounds and enhancing the exploration of microbial secondary metabolites.

Centella asiatica (L.) Urban (Apiaceae) has been utilized for centuries in traditional medicine systems in Southeast Asia and Southern Africa, including Madagascar. Previous studies have reported evidence of the therapeutic potential of C. asiatica formulations in models of Alzheimer’s Disease and other dementias. Caffeoylquinic acids (CQAs) have been identified to be among the pharmacologically relevant metabolites contributing to the botanical’s cognitive enhancement and neuroprotective effects. Isomers of CQAs are, however, difficult to differentiate by commonly used LC-MS techniques, making the characterization, standardization, and batch-to-batch consistency of these formulations challenging. Individual CQAs have unique proton Spin Network Fingerprints (pSNFs) that can be used to distinguish between CQA regioisomers within complex extracts. This work describes the development of a CQA-focused pSNF library that can be used to complement LC-MS methods for the accurate metabolite identification and characterization of bioactive C. asiatica fractions and extracts. The isolation of two new (1 and 2) and four known (3–6) CQAs and CQA analogues from C. asiatica and their contribution to the pSNF library are also discussed herein.

Linalyl anthranilate (LNA) has been identified in a number of plant extracts and essential oils by various authors using gas chromatography–mass spectrometry (GC-MS). However, the reported retention behavior of LNA in these studies is inconsistent with the retention data provided in the NIST database. Therefore, the objective of this study was to determine whether the reports of LNA were the result of misidentifications in GC-MS analyses or if the linear NIST retention index was inaccurate. To accomplish this, linalyl anthranilate was synthesized in a two-step procedure, and the resulting product was authenticated using nuclear magnetic resonance (NMR) and GC-MS analyses. This is a new synthetic route to linalyl anthranilate. Subsequently, retention indices for linalyl anthranilate were determined on three commonly used GC phases: polydimethylsiloxane, 5% diphenyl–95% polydimethylsiloxane, and polyethylene glycol. The study confirmed the accuracy of the NIST retention data, establishing the linear retention index data for LNA on a semi-nonpolar GC column as 2051. However, LNA reported in the literature by various authors exhibited a retention index in the elution window of approximately 1000–1400, strongly suggesting that these reports were the result of GC-MS misidentifications. A review of all reported occurrences of LNA in natural samples found no credible evidence of its presence. In many cases, it appears to be a misidentification of linalyl acetate caused by the occurrence of an erroneous spectrum in the older versions of the NIST mass spectra database.

The virulence-associated isonitrile lipopeptide (INLP) biosynthetic gene cluster is conserved across Mycobacterium tuberculosis and many nontuberculous mycobacteria (NTM) pathogens, yet the corresponding mycobacterial metabolites have not been fully characterized, and their biological functions are still debated. Here, we report a precursor neutral loss chromatography based mass spectrometry strategy that enables the targeted discovery of INLPs from Mycobacterium fortuitum, a fast-growing NTM pathogen. By monitoring a characteristic neutral loss of 27.1 Da corresponding to hydrogen cyanide, we identified a family of INLPs directly from bacterial culture extracts. Structural elucidation of a representative compound using NMR and high-resolution MS revealed a distinctive terminal methylated carboxyl group, contrasting with previously reported INLPs bearing linear alcohol, acetal, or cyclic motifs. Bioinformatic analysis and in vitro enzymatic assays identified a methyltransferase encoded within the INLP BGC responsible for methyl ester formation. Furthermore, metal-binding assays demonstrated selective chelation of Cu(I) and Cu(II) by the isolated INLP, but no detectable interaction with Zn(II), suggesting a role in copper homeostasis. These findings represent the first full structural characterization of an INLP from pathogenic mycobacteria, expand our understanding of the enzymes involved in INLP modification, and unequivocally support the copper-binding activity of INLPs from these pathogens.

Monotropa uniflora L. (“ghost pipe”), is a North American traditional medicinal plant which has recently seen an increase in consumption nationwide. However, a single 1889 study reported the presence of grayanotoxin I in M. uniflora, generating controversy regarding its safety. It is essential to revisit this claim of toxicity given Monotropa uniflora’s resurgence in popularity. We applied more sensitive analytical techniques to determine the possible presence of grayanotoxins in a diverse sampling of M. uniflora, using mass spectrometry and MS2-based molecular networking. Across 50 M. uniflora samples, neither grayanotoxins nor structural analogues were detected, refuting the earlier analysis.