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Funding Information: This publication is based upon work from COST Action CA18238 (Ocean4Biotech), funded by the European Cooperation in Science and Technology (COST) Program in the period 2019–2023. SPG: This work is financed by national funds from FCT—Fundação para a Ciência e a Tecnologia, I.P., in the scope of the project LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy—i4HB. LLB: The publication is part of a project that has received funding from the Erasmus + Project No. ECOBIAS_609967-EPP-1-2019-1-RS-EPPKA2-CBHE-JP; GA.2019-1991/001-001. Development of master curricula in ecological monitoring and aquatic bioassessment for Western Balkans HEIs/ECOBIAS. CJ: This work was supported by grants PID2021-122732OB-C22 from MCIN/AEI/10.13039/501100011033/FEDER “A way to make Europe” (AEI, Spanish State Agency for Research and FEDER Programme from the European Union) and RTI2018-093634-B-C22 from the State Agency for Research (AEI) of Spain, co-funded by the FEDER Programme from the European Union, and BLUEBIOLAB (0474_BLUEBIOLAB_1_E), Programme INTERREG V A of Spain-Portugal (POCTEP). FP: This work is financed by national funds from FCT—Fundação para a Ciência e a Tecnologia, I.P., for an Assistant Research Position (CEECIND/01649/2021). MC: INTERREG-MAC2/1.1b/279 (AHIDAGRO) and the Ministerio de Ciencia e Innovación (Spain) (grant PID2020-115979RR-C32). ARDM is supported with funds from Proyecto Intramural Especial CSIC [Ref. 202280I032]. Publisher Copyright: © 2023 by the authors. Natural Products (NP) are essential for the discovery of novel drugs and products for numerous biotechnological applications. The NP discovery process is expensive and time-consuming, having as major hurdles dereplication (early identification of known compounds) and structure elucidation, particularly the determination of the absolute configuration of metabolites with stereogenic centers. This review comprehensively focuses on recent technological and instrumental advances, highlighting the development of methods that alleviate these obstacles, paving the way for accelerating NP discovery towards biotechnological applications. Herein, we emphasize the most innovative high-throughput tools and methods for advancing bioactivity screening, NP chemical analysis, dereplication, metabolite profiling, metabolomics, genome sequencing and/or genomics approaches, databases, bioinformatics, chemoinformatics, and three-dimensional NP structure elucidation. publishersversion published

Natural Products (NP) are essential for the discovery of novel drugs and products for numerous biotechnological applications. The NP discovery process is expensive and time-consuming, having as major hurdles dereplication (early identification of known compounds) and structure elucidation, particularly the determination of the absolute configuration of metabolites with stereogenic centers. This review comprehensively focuses on recent technological and instrumental advances, highlighting the development of methods that alleviate these obstacles, paving the way for accelerating NP discovery towards biotechnological applications. Herein, we emphasize the most innovative high-throughput tools and methods for advancing bioactivity screening, NP chemical analysis, dereplication, metabolite profiling, metabolomics, genome sequencing and/or genomics approaches, databases, bioinformatics, chemoinformatics, and three-dimensional NP structure elucidation., This publication is based upon work from COST Action CA18238 (Ocean4Biotech), funded by the European Cooperation in Science and Technology (COST) Program in the period 2019–2023. SPG: This work is financed by national funds from FCT—Fundação para a Ciência e a Tecnologia, I.P., in the scope of the projects UIDP/04378/2020 and UIDB/04378/2020 of the Research Unit on Applied Molecular Biosciences—UCIBIO and the project LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy—i4HB. LLB: The publication is part of a project that has received funding from the Erasmus + Project No. ECOBIAS_609967-EPP-1-2019-1-RS-EPPKA2-CBHE-JP; GA.2019-1991/001-001. Development of master curricula in ecological monitoring and aquatic bioassessment for Western Balkans HEIs/ECOBIAS. CJ: This work was supported by grants PID2021-122732OB-C22 from MCIN/AEI/10.13039/501100011033/FEDER “A way to make Europe” (AEI, Spanish State Agency for Research and FEDER Programme from the European Union) and RTI2018-093634-B-C22 from the State Agency for Research (AEI) of Spain, co-funded by the FEDER Programme from the European Union, and BLUEBIOLAB (0474_BLUEBIOLAB_1_E), Programme INTERREG V A of Spain-Portugal (POCTEP). FP: This work is financed by national funds from FCT—Fundação para a Ciência e a Tecnologia, I.P., in the scope of the project UIDB/50006/2020 of the Research Unit on Associated Laboratory for Green Chemistry (LAQV) of the Network of Chemistry and Technology (REQUIMTE) and for an Assistant Research Position (CEECIND/01649/2021). MC: INTERREG-MAC2/1.1b/279 (AHIDAGRO) and the Ministerio de Ciencia e Innovación (Spain) (grant PID2020-115979RR-C32). ARDM is supported with funds from Proyecto Intramural Especial CSIC [Ref. 202280I032].

International audience; Baillonella toxisperma is a medicinal plant used in northern Gabon to treat microbial diseases. It is a plant well-known by local populations, but very few studies have focused on the molecules responsible for the antibacterial activities of B. toxisperma. This study proposes a dereplication strategy based on molecular networking generated from HPLC-ESI-Q/TOF data, allowing investigation of the molecules responsible for the antibacterial activity of B. toxisperma. From this strategy, eighteen compounds were putatively identified. All of these compounds belonged mainly to five families of natural compounds, including phenylpropanolamines, stilbenes, flavonoids, lignans and phenolic glycosides. The chemical study carried out from the bark of B. toxisperma allowed us to identify, for the first time, compounds such as resveratrol and derivatives, epicatechin, epigallocatechin and epigallocatechin gallate. In addition, antibacterial activity (diffusion method and microdilution) and cytotoxicity (Cell Counting Kit-8 (CCK-8 Assay)) in vitro were evaluated. The crude ethanolic extract, as well as the fractions of B. toxisperma, showed significant antibacterial activity. However, the ethanolic fractions F2 and F4 presented high antibacterial activity compared to the crude extract. Cytotoxicity studies on colon-cancer cells (Caco-2) and human keratinocyte cells (HaCaT) showed moderate cytotoxicity in both cell types. This study clearly shows the therapeutic potential of the ethanolic extract of the bark of B. toxisperma and provides information on the phytochemical composition and bioactive compounds of the plant.

The rising incidences of age-related diseases such as Alzheimer and some types of cancer demand novel drugs and therapeutic concepts. Nature has already proven to be an extraordinary rich source of novel structural motifs. They can serve as lead structures for drug development like the prominent antibiotic vancomycin or the anti-cancer drug taxol. In this thesis, a novel natural product named collinolactone was isolated from Streptomyces sp. Gö 40/10. The compound possesses an unprecedented and unique 6-10-7-membered tricyclic system with a cyclodecatriene ring flanked by two lactone rings. Based on stable precursor feeding, a biosynthetic pathway via a type I polyketide synthase was postulated, which was then used for the identification of the encoding gene cluster. Follow-up CRISPR-based genetical engineering, optimization of fermentation parameters and the isolation procedure led to a high-production strain with yields of up to 100 mg/L. A representative and divers set of derivatives of collinolactone was synthesized including the use of Burgess reagent, Strykers reagent and Gilman cuprates. All compounds were profiled in a cell-viability assay on L929 cell line, where some compounds showed cytotoxicity in the micromolar range. The mode of action was studied with fluorescence microscopy on the cell cycle of PtK2 cells and an increased formation of monopolar spindles was observed. The inhibition of putative molecular targets was investigated in a malachite green ATP-based assay. Furthermore, collinolactone was found to exhibit neuroprotective properties in a glutamate induced intramolecular oxidative stress assay on HT22 cells. Only collinolactone showed an effect indicating that the binding is highly specific and even the smallest structural changes will lead to a loss of activity. In addition, the reduction of amyloid beta protein aggregates, one of the hallmarks for the development of Alzheimer disease, was studied. Again, collinolactone showed a reduction of aggregation, making it a very promising lead structure for further development towards a drug for Alzheimer therapy.

Penicillium setosum represents a Penicillium species recently described, with little up-to-date information about its metabolic and biological potential. Due to this scenario, we performed chemical and biological studies of P. setosum CMLD18, a strain isolated from Swinglea glutinosa (Rutaceae). HRMS-MS guided dereplication strategies and anti-leukemia assays conducted the isolation and characterization of six compounds after several chromatographic procedures: 2-chloroemodic acid (2), 2-chloro-1,3,8-trihydroxy-6- (hydroxymethyl)-anthraquinone (7), 7-chloroemodin (8), bisdethiobis(methylthio)acetylaranotine (9), fellutanine C (10), and 4-methyl-5,6-diihydro-2H-pyran-2-one (15). From the assayed metabolites, (10) induced cellular death against Kasumi-1, a human leukemia cell line, as well as good selectivity for it, displaying promising cytotoxic activity. Here, the correct NMR signal assignments for (9) are also described. Therefore, this work highlights more detailed knowledge about the P. setosum chemical profile as well as its biological potential, offering prospects for obtaining natural products with anti-leukemia capabilities.

Chronic liver diseases affect more than 1 billion people worldwide and represent one of the main public health issues. Nonalcoholic fatty liver disease (NAFLD) accounts for the majority of mortal cases, while there is no currently approved therapeutics for its treatment. One of the prospective approaches to NAFLD therapy is to use a mixture of natural compounds. They showed effectiveness in alleviating NAFLD-related conditions including steatosis, fibrosis, etc. However, understanding the mechanism of action of such mixtures is important for their rational application. In this work, we propose a new dereplication workflow for deciphering the mechanism of action of the lignin-derived natural compound mixture. The workflow combines the analysis of molecular components with high-resolution mass spectrometry, selective chemical tagging and deuterium labeling, liver tissue penetration examination, assessment of biological activity in vitro, and computational chemistry tools used to generate putative structural candidates. Molecular docking was used to propose the potential mechanism of action of these structures, which was assessed by a proteomic experiment.

Momordica charantia L. (Cucurbitaceae) is a plant known in Brazil as “melão de São Caetano”, which has been related to many therapeutic applications in folk medicine. Herein, we describe antibacterial activities and related metabolites for an extract and fractions obtained from the leaves of that species. An ethanolic extract and its three fractions were used to perform in vitro antibacterial assays. In addition, liquid chromatography coupled to mass spectrometry and the molecular networking approach were used for the metabolite annotation process. Overall, 25 compounds were annotated in the ethanolic extract from M. charantia leaves, including flavones, terpenes, organic acids, and inositol pyrophosphate derivatives. The ethanolic extract exhibited low activity against Proteus mirabilis (MIC 312.5 µg·mL−1) and Klebsiella pneumoniae (MIC 625 µg·mL−1). The ethyl acetate phase showed interesting antibacterial activity (MIC 156.2 µg·mL−1) against Klebsiella pneumoniae, and it was well justified by the high content of glycosylated flavones. Therefore, based on the ethyl acetate phase antibacterial result, we suggest that M. charantia leaves could be considered as an alternative antibacterial source against K. pneumoniae and can serve as a pillar for future studies as well as pharmacological application against the bacteria.

Background: Lotus maritimus L. (Fabaceae) is a perennial herb species with yellow flowers, growing in Europe, Asia, Middle East and Maghreb. Some flavones and flavonols have been identified in their leaves and flowers. Their leaf extract was used as a cellulolytic and antiaging cosmetic ingredient. Objective: The aim of this work was to perform antioxidant, anti-tyrosinase and anti-elastase properties of L. maritimus aerial parts and their chemical profile. Methods: A 13C NMR-based dereplication method combined with a bio-guided purification was used for metabolite identification. Chemical structures were determined by NMR and ESI-MS spectroscopic methods. The antioxidant properties of the fractions and purified compounds were measured using CUPRAC, DPPH and hydroxyl radical scavenging assays. Their inhibitory activities against the fungal tyrosinase and human neutrophil elastase enzymes were also evaluated. Results: EtOAc and n-BF fractions were characterized as the most active parts. Twenty-two compounds were identified from these fractions by using a 13C NMR-based dereplication method. This process was completed by the purification of minor compounds in the n-BuOH fraction. Eight known compounds were isolated, including many mono-glycosylated flavonoids with variable substitutions on the B-ring, allowing structure-activity relationships. Conclusion: Twenty-two compounds, including phenolic acids, flavonoids and glycoside derivatives, were firstly described in L. maritimus. Three quercetin and myricetin-type flavonoids exhibited good antioxidant activities and all flavonoids tested have moderate effects on elastase inhibition.

Species of the genus Pleiocarpa are used in traditional medicine against fever and malaria. The present study focuses on the isolation and identification of bioactive compounds from P. bicarpellata extracts, and the evaluation of their antiprotozoal activity. Fractionation and isolation combined to LC-HRMS/MS-based dereplication provided 16 compounds: seven indole alkaloids, four indoline alkaloids, two secoiridoid glycosides, two iridoid glycosides, and one phenolic glucoside. One of the quaternary indole alkaloids (7) and one indoline alkaloid (15) have never been reported before. Their structures were elucidated by analysis of spectroscopic data, including 1D and 2D NMR experiments, UV, IR, and HRESIMS data. The absolute configurations were determined by comparison of the experimental and calculated ECD data. The extracts and isolated compounds were evaluated for their antiprotozoal activity towards Trypanosoma brucei rhodesiense, Trypanosoma cruzi, Leishmania donovani, and Plasmodium falciparum, as well as for their cytotoxicity against rat skeletal myoblast L6 cells. The dichloromethane/methanol (1:1) root extract showed strong activity against P. falciparum (IC50 value of 3.5 µg/mL). Among the compounds isolated, tubotaiwine (13) displayed the most significant antiplasmodial activity with an IC50 value of 8.5 µM and a selectivity index of 23.4. Therefore, P. bicarpallata extract can be considered as a source of indole alkaloids with antiplasmodial activity.

Seawater is an environment in which numerous microorganisms have evolved, some with a great potential for biotechnology. In recent years, many scientists have moved away from the assumption that the origin of life was in pools of water, and instead propose that life on Earth probably originated in accumulations of warm, nutrient-rich mud. This mud, also called peloid is a rich source of organisms that, due to their adaptation to this unique environment, produce a wide variety of primary and secondary metabolites with numerous and diverse activities, including anti-cancer, anti-inflammatory, antiviral, and immunomodulatory ones. In this research, two questions were addressed using collected samples of a peloid with demonstrated healing properties. Firstly biodiversity in this ecological niche was explored in order to assess microbial communities present and secondly natural products were screened for in order to assess whether predicted activities could be linked to healing properties of the peloid. The use of peloids in medical therapy dates back to ancient times. Abiotic components such as clay and mineral water are believed to be the main contributors of the healing properties of natural peloids. The places where peloids are usually found are characteristically shallow and enclosed lagoons. The constant UV exposure and increased salt concentration classify peloid as an extreme environment. The spectrum of relief’s peloid therapy is claimed to provide ranges from purely cosmetic and skin-related to musculoskeletal and immunological problems. These claims can hardly be supported by mineral content and heat-retaining properties alone. However, organic compounds from present microorganisms as well as secondary metabolites could help explain the observed range of health benefits. The fact that the relationship between the therapeutic activity of peloids and their composition besides mineral and physicochemical properties has not been extensively studied indicates untapped biotechnological potential.

The genus Banisteriopsis (Malpighiaceae) comprises 77 genera and 1,300 species. Despite efforts to provide detailed information about the chemical wealth of B. laevifolia, this article provides the identification and characterization of compounds from the ethanolic extracts of the leaves and flowers using advanced methodologies which include high-performance liquid chromatography coupled to tandem mass spectrometry, which contribute to the knowledge about compounds present in the genus Banisteriopsis. The dereplication aided by Global Natural Products Social Molecular Networking afforded the identification of seven compounds reported for this species for the first time. A mixture of two known flavonoids and a diterpenoid, 18-hydroxy-ent-halima-1(10),13-(E)-dien-15-oic acid, were isolated by conventional separation methods. The elevated 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity observed in some samples was attributed to either the type of extract, the presence of 3,4,5-trihydroxybenzoic acid, or the influence of the substitution pattern and the synergistic effect of the compounds present. Principal Component Analysis was applied to correlate mass spectrometry data with the antioxidant activity of the samples. The high diversity of metabolites found in this study and those which have been reported for Banisteriopsis strongly recommends further investigation into the chemical and biological properties of these species.

Dunaliella tertiolecta is a green flagellated microalga with a high tolerance to salinity and high production of pigments such as zeaxanthin, a xanthophyll carotenoid present in higher plants known for its antioxidant potential. In the current study, the antiproliferative activity of raw extracts and fractions of D. tertiolecta (clone CCMP 1320) was evaluated against four different human cancer cell lines: melanoma, hepatocellular liver carcinoma, and two lung adenocarcinoma cell lines. In addition, a normal cell line (lung fibroblast) was used as toxicity control. The activity was evaluated by treatment with the extracts/fractions following the MTT colorimetric assay procedures. HPLC-UV-HRMS based dereplication helped to identify the bioactive metabolites. A glycoglycerolipid was identified in the active fraction, being involved in the bioactivity of this microalga. This compound, glycerol 1-(9Z,12Z,15Z-octadecatrienoate)-2-(4Z,7Z,10Z,13Z-hexadecatetraenoate)-3-O-β-D-galactopyranoside (1), was purified and its antiproliferative activity was confirmed. This work gives new insights on the antiproliferative activity of the green alga D. tertiolecta and its potential industrial applications.

Lack of new anti-cancer and anti-infective agents directed the pharmaceutical research to natural products' discovery especially from actinomycetes as one of the major sources of bioactive compounds. Metabolomics- and dereplication-guided approach has been used successfully in chemical profiling of bioactive actinomycetes. We aimed to study the metabolomic profile of five bioactive actinomycetes to investigate the interesting metabolites responsible for their antimicrobial and anti-cancer activities. Three actinomycetes, namely, Streptomyces sp. SH8, SH10 and SH13, were found to exhibit broad spectrum of antimicrobial activities, whereas isolate SH4 showed the broadest antimicrobial activity against all tested strains. In addition, isolates SH8, SH10 and SH12 displayed potent cytotoxicity against the breast cancer cell line Michigan Cancer Foundation-7 (MCF-7), whereas isolates SH4 and SH12 exhibited potent anti-cancer activity against the hepatoma cell line hepatoma G2 (HepG2) compared with their weak inhibitory properties on the normal breast cells MCF-10A and normal liver cells transformed human liver epithelial-2 (THLE2), respectively. All bioactive isolates were molecularly identified as Streptomyces sp. via 16S rRNA gene sequencing. Our actinobacterial dereplication analysis revealed putative identification of several bioactive metabolites including tetracycline, oxytetracycline and a macrolide antibiotic, novamethymycin. Together, chemical profiling of bioactive Streptomycetes via dereplication and metabolomics helped in assigning their unique metabolites and predicting the bioactive compounds instigating their diverse bioactivities.

Despite considerable advances in medicine and technology, humanity still faces many deadly diseases such as cancer and malaria. In order to find appropriate treatments, the discovery of new bioactive substances is essential. Therefore, research is now turning to less frequently explored habitats with exceptional biodiversity such as the marine environment. Many studies have demonstrated the therapeutic potential of bioactive compounds from marine macro- and microorganisms. In this study, nine microbial strains isolated from an Indian Ocean sponge, Scopalina hapalia, were screened for their chemical potential. The isolates belong to different phyla, some of which are already known for their production of secondary metabolites, such as the actinobacteria. This article aims at describing the selection method used to identify the most promising microorganisms in the field of active metabolites production. The method is based on the combination of their biological and chemical screening, coupled with the use of bioinformatic tools. The dereplication of microbial extracts and the creation of a molecular network revealed the presence of known bioactive molecules such as staurosporin, erythromycin and chaetoglobosins. Molecular network exploration indicated the possible presence of novel compounds in clusters of interest. The biological activities targeted in the study were cytotoxicity against the HCT-116 and MDA-MB-231 cell lines and antiplasmodial activity against Plasmodium falciparum 3D7. Chaetomium globosum SH-123 and Salinispora arenicola SH-78 strains actually showed remarkable cytotoxic and antiplasmodial activities, while Micromonospora fluostatini SH-82 demonstrated promising antiplasmodial effects. The ranking of the microorganisms as a result of the different screening steps allowed the selection of a promising strain, Micromonospora fluostatini SH-82, as a premium candidate for the discovery of new drugs.

The structure elucidation of small organic molecules (

The detailed assessment of the composition of plant-derived products is of primary interest. The metabolites in natural extracts (NE) constitute the metabolome, which can be divided into the core and the specialized metabolome. Plants produce specialized metabolites to ensure their survival in a competitive environment. To assess the composition of NEs, currently validated methods for rigorous annotation and quantification of metabolites require standards. However, among the known metabolites, the availability of commercial reference standards is heavily restricted. Given this limitation, common analytical methods for NE composition assessment focus on studying a few specific and often non-bioactive markers. Liquid chromatography coupled to mass spectrometry (LC-MS) is a method of choice for NE metabolite analysis. Annotating data sets generated by LC-MS systems remains challenging. Dereplication allows focusing efforts on novel compounds, overcoming this challenge by leveraging prior knowledge and computational tools. In the frame of the present thesis, two resources to improve dereplication were developed. The first is the Taxonomically Informed Metabolite Annotation, which allows for better decision-making when multiple structural candidates are suggested by current MS-based annotation tools. The second is LOTUS, an initiative for open knowledge management in natural products research, that provides the largest collection of metabolite-taxon pairs. In addition to annotation, semi-quantitative aspects are crucial for NE composition evaluation. They are needed to document the use of NEs as products and assess the presence and concentration level of potentially toxic compounds. Such information may also provide a rationale to justify specific molecules’ contribution to an extract’s overall bioactivity. Nevertheless, generic methods generating a semi-quantitative assessment of a large panel of metabolites are still lacking. Typically, only a dozen metabolites account for most of the extract’s mass, while hundreds are present in trace amounts. Therefore, effective procedures providing a comprehensive analysis of the metabolome of NEs are needed, further addressing both qualitative and quantitative aspects. This work combines qualitative and semi-quantitative information in an automated manner, by integrating LC-MS-based metabolite profiling with generic universal detection methods. The impact of this strategy is evaluated on public data, collaborations, and well-known plants. Its application to different research questions is illustrated, i.e. through flavoring plants of industrial interest such as Swertia chirayita (Roxb.) H. Karst, containing large quantities of bitter principles. The presented workflow, integrating analytical and computational strategies, aims to make plant metabolomics research more effective for public health, food and beverage safety, as well as fundamental science., L'évaluation détaillée de la composition des produits dérivés des plantes est d'intérêt primordial. Les métabolites présents dans les extraits naturels (NE) constituent le métabolome, qui peut être divisé en métabolome principal et métabolome spécialisé. Les plantes produisent des métabolites spécialisés pour assurer leur survie dans un environnement compétitif. Pour évaluer la composition des NE, les méthodes actuellement validées pour l'annotation et la quantification rigoureuses des métabolites nécessitent des standards. Cependant, parmi les métabolites connus, la disponibilité de standards de référence commerciaux est fortement limitée. Compte tenu de cette limitation, les méthodes analytiques courantes pour l'évaluation de la composition du NE se fondent sur l'étude de quelques marqueurs spécifiques et souvent non bioactifs. La chromatographie liquide couplée à la spectrométrie de masse (LC-MS) est une méthode de choix pour l'analyse des métabolites de NE. L'annotation des données générées par ces systèmes LC-MS reste un défi. La déréplication permet de concentrer les efforts sur des composés nouveaux, surmontant ce défi en tirant parti des connaissances antérieures et des outils informatiques. Dans le cadre de la présente thèse, deux ressources visant à améliorer la déréplication ont été développées. La première est l'Annotation de Metabolites Informée Taxonomiquement, qui permet une meilleure prise de décision lorsque de multiples candidats structurels sont suggérés par les outils d'annotation actuels basés sur la MS. Le second est LOTUS, une initiative pour la gestion ouverte des connaissances dans la recherche sur les produits naturels, qui fournit la plus grande collection de paires métabolite-taxon. Outre l'annotation, les aspects semi-quantitatifs sont cruciaux pour l'évaluation de la composition des NE. Ils sont nécessaires pour documenter l'utilisation des NE en tant que produits et évaluer la présence et le niveau de concentration de composés potentiellement toxiques. Ces informations peuvent également permettre de justifier la contribution de molécules spécifiques à la bioactivité globale d'un extrait. Néanmoins, il n'existe toujours pas de méthodes génériques permettant une évaluation semi-quantitative d'un large panel de métabolites. Typiquement, seule une douzaine de métabolites représentent la majeure partie de la masse de l'extrait, tandis que des centaines sont présents à l'état de traces. Il est donc nécessaire de mettre au point des procédures efficaces permettant une analyse complète du métabolome des NE, en tenant compte des aspects qualitatifs et quantitatifs. Ce travail combine des informations qualitatives et semi-quantitatives de manière automatisée, en intégrant le profilage des métabolites par LC-MS avec des méthodes de détection universelles génériques. L'impact de cette stratégie est évalué sur des données publiques, des collaborations et différentes plantes connues. Son application à différentes questions de recherche est illustrée, par exemple à travers des plantes aromatiques ou sapides d'intérêt industriel telles que Swertia chirayita (Roxb.) H. Karst, contenant de grandes quantités de principes amers. Le flux de travail présenté, intégrant des stratégies analytiques et computationnelles, vise à rendre la recherche en métabolomique végétale plus efficace pour la santé publique, la sécurité des aliments et des boissons, ainsi que pour la science fondamentale., Die detaillierte Bewertung der Zusammensetzung von Pflanzenprodukten ist von vorrangigem Interesse. Die Metaboliten in den natürlichen Extrakten (NE) bilden das Metabolom, das in Hauptmetabolom und spezialisiertes Metabolom unterteilt werden kann. Pflanzen produzieren spezialisierte Metaboliten, um ihr Überleben in einer kompetitiven Umgebung zu sichern. Um die Zusammensetzung der NE zu bewerten, sind Standards für die derzeit validierten Methoden zur strengen Annotation und Quantifizierung von Metaboliten erforderlich. Unter den bekannten Metaboliten ist die Verfügbarkeit von kommerziellen Referenzstandards jedoch stark eingeschränkt. Angesichts dieser Einschränkung basieren die gängigen Analysemethoden zur Bewertung der NE-Zusammensetzung auf der Untersuchung einiger weniger spezifischer und oft nicht bioaktiver Marker. Die Flüssigchromatographie gekoppelt mit Massenspektrometrie (LC-MS) ist eine Methode der Wahl für die Analyse von NE-Metaboliten. Die Annotation der Datensätze, die von LC-MS-Systemen erzeugt werden, bleibt eine Herausforderung. Die Dereplikation ermöglicht den Fokus auf neue Verbindungen und überwindet diese Herausforderung durch die Nutzung von Vorwissen und Computerwerkzeugen. Im Rahmen der vorliegenden Dissertation wurden zwei Ressourcen zur Verbesserung der Dereplikation entwickelt. Die erste ist die Taxonomically Informed Metabolite Annotation, die eine bessere Entscheidungsfindung ermöglicht, wenn mehrere strukturelle Kandidaten von den aktuellen MS-basierten Annotationswerkzeugen vorgeschlagen werden. Das zweite ist LOTUS, eine Initiative für offenes Wissensmanagement in der Naturstoffforschung, die die größte Sammlung von Metabolit-Taxon-Paaren zur Verfügung stellt. Neben der Annotation sind semiquantitative Aspekte für die Bewertung der Zusammensetzung von NE von entscheidender Bedeutung. Sie sind notwendig, um die Verwendung von NE als Produkte zu dokumentieren und das Vorhandensein und die Konzentrationshöhe potenziell toxischer Verbindungen zu bewerten. Diese Informationen können auch dazu dienen, den Beitrag spezifischer Moleküle zur Gesamtbioaktivität eines Extrakts zu begründen. Dennoch gibt es immer noch keine generischen Methoden, die eine semiquantitative Bewertung eines breiten Spektrums von Metaboliten ermöglichen. Typischerweise machen nur ein Dutzend Metaboliten den größten Teil der Masse des Extrakts aus, während Hunderte in Spuren vorhanden sind. Daher müssen effiziente Verfahren entwickelt werden, die eine umfassende Analyse des Metaboloms von NE unter Berücksichtigung qualitativer und quantitativer Aspekte ermöglichen. In dieser Arbeit werden qualitative und semiquantitative Informationen auf automatisierte Weise kombiniert, indem das LC-MS-basierte Metabolitenprofiling mit allgemeinen universellen Nachweismethoden integriert wird. Die Auswirkungen dieser Strategie werden anhand von öffentlichen Daten, Kollaborationen und verschiedenen bekannten Pflanzen. Ihre Anwendung auf verschiedene Forschungsfragen wird veranschaulicht, z. B. anhand von aromatischen oder sapiden Pflanzen von industriellem Interesse wie Swertia chirayita (Roxb.) H. Karst, die große Mengen an Bitterstoffen enthalten. Der vorgestellte Arbeitsablauf, der analytische und computergestützte Strategien integriert, soll die Forschung im Bereich Pflanzenmetabolomik für die öffentliche Gesundheit, die Lebensmittel- und Getränkesicherheit sowie die Grundlagenwissenschaft effizienter machen., La valutazione dettagliata della composizione dei prodotti di origine vegetale è di interesse primario. I metaboliti presenti negli estratti naturali (NE) costituiscono il metaboloma, che può essere suddiviso in metaboloma principale e metaboloma specializzato. Le piante producono metaboliti specializzati per garantire la loro sopravvivenza in un ambiente competitivo. Per valutare la composizione dei NE, i metodi attualmente validati per l'annotazione e la quantificazione rigorosi dei metaboliti richiedono degli standard. Tuttavia, dei metaboliti conosciuti, la disponibilità di standard commerciali di riferimento è fortemente limitata. Data questa limitazione, gli metodi analitici attuali per la valutazione della composizione dei NE si basano sullo studio di pochi marcatori specifici e spesso non bioattivi. La cromatografia liquida-spettrometria di massa (LC-MS) è un metodo di scelta per l'analisi dei metaboliti dei NE. L'annotazione degli dati generati da LC-MS rimane una sfida. La dereplicazione consente di concentrare gli sforzi su composti nuovi, superando questa sfida sfruttando le conoscenze pregresse e i strumenti computazionali. Nel contesto di questa tesi, sono state sviluppate due risorse per migliorare la dereplicazione. Il primo è la Taxonomically Informed Metabolite Annotation, che consente di prendere decisioni migliori quando gli attuali strumenti di annotazione basati sulla MS suggeriscono più candidati strutturali. Il secondo è LOTUS, un'iniziativa per la gestione aperta della conoscenza nella ricerca sui prodotti naturali, che fornisce la più ampia raccolta di coppie metabolita-tassone. Oltre all'annotazione, gli aspetti semiquantitativi sono fondamentali per la valutazione della composizione dei NE. Essi sono necessari per documentare l'uso dei NE come prodotti e per valutare la presenza e il livello di concentrazione di composti potenzialmente tossici. Queste informazioni possono essere utilizzate anche per giustificare il contributo di specifiche molecole alla bioattività complessiva di un estratto. Tuttavia, non esistono ancora metodi generici per la valutazione semiquantitativa di un'ampia gamma di metaboliti. In genere, solo una decina di metaboliti costituiscono la maggior parte dell'estratto, mentre centinaia sono presenti in tracce. È quindi necessario sviluppare procedure efficienti per un'analisi completa del metaboloma dei NE, che tenga conto di aspetti sia qualitativi che quantitativi. Questo lavoro combina informazioni qualitative e semi-quantitative in modo automatizzato, integrando la profilazione dei metaboliti basata su LC-MS con metodi generici di rilevazione universale. L'impatto di questa strategia è valutato su dati pubblici, collaborazioni e diverse piante note. La sua applicazione a diverse questioni di ricerca è illustrata, ad esempio attraverso piante aromatiche o sapide di interesse industriale come la Swertia chirayita (Roxb.) H. Karst, contenente grandi quantità di principi amari. Il flusso di lavoro presentato, che integra strategie analitiche e computazionali, mira a rendere più efficace la ricerca sulla metabolomica vegetale per la salute pubblica, la sicurezza di alimenti e bevande e la scienza di base.

Cyclic lipopeptides (CLiPs) are secondary metabolites secreted by a range of bacterial phyla. CLiPs from Pseudomonas in particular, display diverse structural variations in terms of the number of amino acid residues, macrocycle size, amino acid identity, and stereochemistry (e.g., d- versus l-amino acids). Reports detailing the discovery of novel or already characterized CLiPs from new sources appear regularly in literature. Increasingly, however, the lack of detailed characterization threatens to cause considerable confusion, especially if configurational heterogeneity is present for one or more amino acids. Using Pseudomonas CLiPs from the Bananamide, Orfamide, and Xantholysin groups as test cases, we demonstrate and validate that the combined 1H and 13C Nuclear Magnetic Resonance (NMR) chemical shifts of CLiPs constitute a spectral fingerprint that is sufficiently sensitive to differentiate between possible diastereomers of a particular sequence even when they only differ in a single d/l configuration. Rapid screening, involving simple matching of the NMR fingerprint of a newly isolated CLiP with that of a reference CLiP of known stereochemistry, can then be applied to resolve dead-ends in configurational characterization and avoid the much more cumbersome chemical characterization protocols. Even when the stereochemistry of a particular reference CLiP remains to be established, its spectral fingerprint allows to quickly verify whether a newly isolated CLiP is novel or already present in the reference collection. We show NMR fingerprinting leads to a simple approach for early on dereplication which should become more effective as more fingerprints are collected. To benefit research involving CLiPs, we have made a publicly available data repository accompanied by a 'knowledge base' at https://www.rhizoclip.be, where we present an overview of published NMR fingerprint data of characterized CLiPs, together with literature data on the originally determined structures. IMPORTANCE Pseudomonas CLiPs are ubiquitous specialized metabolites, impacting the producer's lifestyle and interactions with the (a)biotic environment. Consequently, they generate interest for agricultural and clinical applications. Establishing structure-activity relationships as a premise to their development is hindered because full structural characterization including stereochemical information requires labor-intensive analyses, without guarantee for success. Moreover, increasing use of superficial comparison with previously characterized CLiPs introduces or propagates erroneous attributions, clouding further scientific progress. We provide a generally applicable characterization methodology based on matching NMR spectral fingerprints of newly isolated CLiPs to natural and synthetic reference compounds with (un)known stereochemistry. In addition, NMR fingerprinting is shown to provide a suitable basis for structural dereplication. A publicly available reference compound repository promises to facilitate participation of the lipopeptide research community in structural assessment and dereplication of newly isolated CLiPs, which should also support further developments in genome mining for novel CLiPs. ispartof: MICROBIOLOGY SPECTRUM vol:10 issue:4 ispartof: location:United States status: published

ilustraciones, fotografías a color El uso de organismos biocontroladores como estrategia que permita disminuir el uso de agroquímicos en los cultivos, es una práctica que está en aumento. Particularmente se hace relevante el desarrollo de productos de origen biológico que permitan el control de patógenos de arroz de manera específica y eficaz disminuyendo las tasas de contaminación y la reincidencia de enfermedades por la aparición de patógenos resistentes. Sin embargo, para llegar al desarrollo de un producto de origen biológico que tenga las características necesarias para ser efectivo en el control de enfermados fitosanitarias, es necesario tener un conocimiento amplio del organismo productor de los principios activos que harán parte de dicho producto. Bajo este contexto, el estudio de la cepa Bacillus velezensis IBUN 2755 se hace relevante ya que ha demostrado tener una fuerte actividad antibacteriana y antifúngica in vitro, contra tres patógenos que han sido reportados como los causantes de grandes pérdidas en cultivos de arroz y por tanto esta cepa se perfila como un potencial microorganismo biocontrolador. El estudio extensivo de la cepa incluye la caracterización de los posibles metabolitos secundarios bioactivos que produzca la cepa, que estén involucrados de manera directa en la actividad antibacterial y antifúngica conocida, asi como el efecto que tienen diferentes factores bióticos y abióticos sobre su producción. Este es precisamente el objetivo principal de este trabajo de investigación. El primer paso para llegar a este objetivo fue describir el efecto que tiene el crecimiento de la cepa IBUN 2755 en diferentes medios de cultivo, sobre la actividad antagónica de la cepa contra los patógenos Burkholderia. glumae, Rhizoctonia solani y Gaeumannomyces graminis. Para esto, se incubó la cepa IBUN 2755 en los medios de cultivo Landy, MES, MES modificado y MOLP P.E y MOLP P.U, que se caracterizan por tener condiciones nutricionales diferenciales. Se recuperaron los sobrenadantes libres de células de cada medio posterior al crecimiento de la cepa y se evaluó la actividad antagónica de los mismos sobre los tres patógenos antes mencionados. Se pudo concluir, que la composición nutricional de los medios tiene un efecto directo sobre la actividad antagónica de la cepa IBUN 2755, siendo el medio MES el que presentó sobrenadantes menos activos contra los tres patógenos y el medio MOLP P.U el que rindió los sobrenadantes más activos. Posteriormente con el fin de evaluar los metabolitos bioactivos que se estaban expresando en estos dos medios de cultivo que mostraron tener una actividad antagónica diferencial (MOLP.U y MES), se decidió hacer la extracción de estos usando dos metodologías de extracción: la partición liquido - liquido usando acetato de etilo y butanol y la precipitación ácida. Asi mismo estos procesos de extracción se realizaron sobre dos volúmenes diferentes de cada medio: 25 y 200 ml. De esta forma, al finalizar esta sección se tendrían tres parámetros a evaluar: primero, el efecto de la composición nutricional del medio de cultivo sobre la producción de metabolitos secundarios, segundo el efecto de la metodología de extracción sobre los metabolitos bioactivos recuperados de cada medio y tercero el efecto del volumen del medio de cultivo sobre la producción de metabolitos secundarios bioactivos por parte de la cepa IBUN 2755. La evaluación de la actividad biológica de los extractos de acetato de etilo, butanol y precipitación ácida obtenidos a partir de los sobrenadantes recuperados de los medios MOLP P.U y MES de 200 y 25 ml, sobre los tres patógenos, mostró que el extracto por precipitación acida del medio MOLP P.U de 25 ml, era el que demostraba una actividad antagónica consistente contra los tres patógenos. Asi mismo, se caracterizó el perfil metabolómico de cada uno estos extractos usando LC-MS/MS la construcción de redes moleculares y la derreplicación de compuestos, con esto se pudo establecer que la expresión de metabolitos secundarios por parte de la cepa IBUN 2755, era mayor en los medios de 25ml para el caso del medio MOLP P.U, además se evidencio que los extractos por precipitación acida y de acetato de etilo eran los más enriquecidos en compuestos con respecto a los extractos butanólicos y finalmente, se logró establecer que si bien la cepa IBUN 2755, es capaz de producir las mismas familias de compuestos, en los dos medios de cultivo (MOLP P.U y MES) esto no implica que se produzcan los mismos compuestos y en la misma proporción. Así mismo se evidencio la expresión de moléculas de interés como surfactinas, fengicinas e Iturinas. Una vez establecido el efecto de diferentes factores sobre la producción de metabolitos secundarios por parte de la cepa IBUN 2755, el siguiente paso fue identificar los compuestos responsables de la actividad antagónica de la cepa contra los patógenos B. glumae, R. solani y G. graminis, para esto se fracciono el extracto por precipitación con ácido del medio MOLP P.U y se evaluó la actividad de cada una de las fracciones, las fracciones activas fueron evaluadas por RMN y LC-MS/MS. Las fracciones F4 y F5 mostraron ser las más activas contra los tres patógenos y se logró determinar que los compuestos activos mayoritarios de estas fracciones eran la Surfactina C14 ß-OH ácido graso-MeO-Glu-Leu-Leu-Val-Asp-Leu-Leu y la surfactina C14 ß-OH ácido graso-Glu-Leu-Leu-Val-Asp-Leu-Leu. Finalmente, se decidió caracterizar de manera descriptiva haciendo uso de redes moleculares, el perfil metabolómico de la cepa IBUN 2755 bajo condiciones de cocoltivo con cada uno de los patógenos evaluados en este proyecto, con el fin de evaluar el efecto del crecimiento de la cepa en conjunto con otro microorganismo, sobre la producción de metabolitos secundarios potencialmente bioactivos. Este análisis metabolómico incluyo datos de LC-MS/MS tanto de los monocultivos de cada microorganismo, como de los cocultivos entre la cepa biocontroladora y cada una de los patógenos. Los resultados mostraron que en efecto cuando la cepa se encuentra bajo condiciones de estrés, como lo es enfrentarse a otro microorganismo, la expresión de metabolitos secundarios bioactivos se eleva drásticamente y se difunden hacia el área de crecimiento del patógeno, esto es especialmente cierto para los cocultivos de la cepa IBUN 2755 - B. glumae y IBUN 2755 - G. graminis. (Texto tomado de la fuente) The use of biocontrol organisms as a strategy to reduce the use of agrochemicals in crops is a practice that is on the rise. Particularly relevant is the development of products of biological origin that allow the control of rice pathogens in a specific and effective way, reducing contamination rates and the recurrence of diseases due to the appearance of resistant pathogens. However, to develop a product of biological origin that has the necessary characteristics to be effective in controlling phytosanitary diseases, it is necessary to have extensive knowledge of the organism that produces the active ingredients that will be part of said product. In this context, the study of the Bacillus velezensis IBUN 2755 strain becomes relevant since it has been shown to have a strong antibacterial and antifungal activity in vitro, against three pathogens that have been reported as the cause of great losses in rice crops and therefore this strain is outlined as a potential biocontrol microorganism The extensive study of the strain includes the characterization of the possible bioactive secondary metabolites produced by the strain, which are directly involved in the known antibacterial and antifungal activity, as well as the effect that different biotic and abiotic factors have on its production. This is precisely the main objective of this research work. The first step to reaching this goal was to describe the effect of the growth of the IBUN 2755 strain in different culture media on the antagonistic activity of the strain against the pathogens B. glumae, R. solani and G. graminis. For this, the IBUN 2755 strain was incubated in Landy, MES, modified MES and MOLP P.E, and MOLP P.U culture media, which are characterized by having differential nutritional conditions. Cell-free supernatants were recovered from each medium after the growth of the strain and their antagonistic activity on the three aforementioned pathogens was evaluated. It was concluded that the nutritional composition of the media has a direct effect on the antagonistic activity of the IBUN 2755 strain, with the MES medium being the one that presented less active supernatants against the three pathogens and the MOLP P.U medium the one that yielded the most active supernatants. assets. Subsequently, to evaluate the bioactive metabolites that were being expressed in these two culture media that showed a differential antagonistic activity (MOLP.U and MES), it was decided to extract them using two extraction methodologies: liquid partition - liquid using ethyl acetate and butanol and acid precipitation. Likewise, these extraction processes were carried out on two different volumes of each medium: 25 and 200 ml. In this way, at the end of this section there would be three parameters to evaluate: first, the effect of the nutritional composition of the culture medium on the expression of secondary metabolites, second, the effect of the extraction methodology on the bioactive metabolites recovered from each medium and third, the effect of the volume of the culture medium on the production of bioactive secondary metabolites by the IBUN 2755 strain The evaluation of the biological activity of the extracts of ethyl acetate, butanol, and acid precipitation obtained from the supernatants recovered from the MOLP P.U and MES media of 200 and 25 ml, on the three pathogens, showed that the extract by acid precipitation of the 25ml MOLP P.U medium was the one that demonstrated consistent antagonistic activity against the three pathogens. Likewise, the metabolic profile of each of these extracts was characterized using LC-MS/MS, the construction of molecular networks, and the dereplication of compounds, with this it was possible to establish that the expression of secondary metabolites by the IBUN 2755 strain was higher in the 25ml media for the case of the MOLP P.U medium, it was also evidenced that the extracts by acid precipitation and ethyl acetate were the most enriched in compounds concerning the butanol extracts and finally, it was possible to establish that although the IBUN 2755 strain, is capable of expressing the same families of compounds, in the two culture media (MOLP P.U and MES) this does not imply that the same compounds are expressed and in the same proportion. Likewise, the expression of molecules of interest such as surfactins, fengycins and Iturins was evidenced. Once the effect of different factors on the production of secondary etabolites by the IBUN 2755 strain had been established, the next step was to identify the compounds responsible for the antagonistic activity of the strain against the pathogens B. glumae, R. solani and G. graminis, for this, the extract was fractionated by acid precipitation of the MOLP P.U medium and the activity of each of the fractions was evaluated, the active fractions were evaluated by NMR and LC-MS/MS. Fractions F4 and F5 showed to be the most active against the three pathogens and it was possible to determine that the main active compounds of these fractions were Surfactin C14 ß-OH fatty acid-MeO-Glu-Leu-Leu-Val-Asp-Leu- Leu and surfactin C14 ß-OH fatty acid-Glu-Leu-Leu-Val-Asp-Leu-Leu. Finally, it was decided to characterize descriptively, using molecular networks, the metabolic profile of the IBUN 2755 strain under co-culture conditions with each of the pathogens evaluated in this project, to evaluate the effect of the growth of the strain on together with another microorganism, on the expression of potentially bioactive secondary metabolites. This metabolomic analysis included LC-MS/MS data from both the monocultures of each microorganism and the co-cultures between the biocontrol strain and each of the pathogens. The results showed that, in effect, when the strain is under stress conditions, such as facing another microorganism, the expression of bioactive secondary metabolites rises drastically and diffuses towards the area of growth of the pathogen, this is especially true for microorganisms. cocultures of strain IBUN 2755 - B. glumae and IBUN 2755 - G. graminis. Maestría Magíster en Ciencias - Microbiología Metabolomica

Three endophytic fungi isolated from Moquiniastrum polymorphum (Less.) G. Sancho (Asteraceae) were cultivated using the one strain many compounds (OSMAC) strategy to evaluate the production of griseofulvin derivatives. Extracts obtained were analyzed by HPLC–MS/MS and the chromatographic and spectrometric data used to elaborate a feature-based molecular network (FBMN) through the GNPS platform. This approach allowed the observation of differences such as medium-specific and strain-specific production of griseofulvin derivatives and variations of cytotoxic activity in most extracts. To evaluate the efficiency of the OSMAC approach allied with FBMN analysis in the prospection of compounds of biotechnological interest, griseofulvin and 7-dechlorogriseofulvin were isolated, and the relative concentrations were estimated in all culture media using HPLC–UV, allowing for the inference of the best strain–medium combinations to maximize its production. Malt extract-peptone broth and Wickerham broth media produced the highest concentrations of both secondary metabolites.

Phytochemical investigation of the dichloromethane crude extract from the bark of Ochreinauclea maingayi with the aid of LCMS/MS-based molecular networking guided the isolation and accelerated the elucidation of known and new indole alkaloids. The molecular networking analysis produces two main clusters, along with 41 non-prioritized clusters and self-loop nodes. Each cluster has several nodes which depict the fractions contained within those nodes. An implementation of a fraction mapping for each node represents the molecular weight and key fragment data of each compound. From the analysis of each cluster and node, we can deduce the indole alkaloids are the scaffold of interest. Indole scaffold can be found between F5 and F10 that contain several types of indole alkaloids. In total, we have successfully purified nine indole alkaloids, including 9H-β-carboline-4-carboxylate 2, norharmane 3, harmane 4, naucledine 10, neonaucline 15, 1,2,3,4-tetranorharmane-1-one 16, naulafine 19, cadambine 9, and a new monoterpene indole alkaloid dihyrodeglycocadambine 7 from F5 to F10 using a chromatographic technique. Their structures were confirmed by 1D-NMR, 2D-NMR, UV, IR, LCMS, and MS2LDA. Several clusters and nodes contain ions that could not be annotated, suggesting that they may possess novel compounds that are yet to be discovered.

Restingas are extreme environments present in the Atlantic Rainforest biome. These ecosystems show peculiar characteristics, like sandy soil, high salinity, and high solar exposure, which brings scientific interest to their native species. Stachytarpheta schottiana is an endemic Brazilian species found in Jurubatiba Restinga, and just like other species of the genus Stachytarpheta, is used in folk medicine. In this paper, we describe, for the first time, 28 secondary metabolites from S. schottiana polar extract, among them iridoids, flavonoids, lignans and phenylethanoids, with the use of LC-HRMS/MS dereplication and molecular networking methodologies. Many of these compounds have not been described yet for the genus Stachytarpheta, like astragalin, taxifolin, lamiide and the lignans secondary metabolites class. Also, in this paper, High Speed Counter-Current Chromatography (HSCCC) isolation and Nuclear Magnetic Resonance (NMR) of two compounds were used to correct a misidentification in the dereplication procedure and to create seeds for molecular networking. Some of the suggested known compounds found in this work have had their biological activities described in the literature, such as the hepatoprotective activity of verbascoside, that matches those already related for the genus Stachytarpheta and for the folk use of Stachytarpheta schottiana itself. Resumo As restingas são ambientes com características extremas, que fazem parte do bioma da Mata Atlântica. Estes ambientes apresentam características peculiares, como solo arenoso e altas salinidade e exposição solar, o que torna as espécies presentes interessantes para a ciência. A espécie Stachytarpheta schottiana é endêmica do Brasil, presente em restingas e, assim como outras espécies do gênero, apresenta uso na medicina popular. Neste trabalho são descritos pela primeira vez 28 metabólitos secundários da espécie S. schottiana, como iridoides, lignanas, flavonoides e feniletanoides, através do uso de metodologias de desreplicação e molecular networking por LC-HRMS/MS. Destas substâncias, muitas sequer foram descritas para o gênero, como astragalina, taxifolina, lamiídeo e toda a classe de lignanas. Ainda neste trabalho, a Cromatografia Contracorrente de Alta Velocidade (High Speed Countercurrent Chromatography - HSCCC) e a Ressonância Magnética Nuclear (RMN) foram utilizadas para corrigir a identificação equivocada de duas substâncias, bem como para a criação de seeds que seriam posteriormente utilizadas nos molecular networkings. Algumas das substâncias conhecidas sugeridas neste trabalho apresentam atividades biológicas já descritas na literatura que coincidem com o uso popular descrito para a espécie e também para o gênero, como o efeito hepatoprotetor do verbascosídeo.

Since the golden age of antibiotics in the 1950s and 1960s actinomycetes have been the most prolific source for bioactive natural products. However, the number of discoveries of new bioactive compounds decreases since decades. New procedures (e.g., activating strategies or innovative fermentation techniques) were developed to enhance the productivity of actinomycetes. Nevertheless, compound identification remains challenging among others due to high rediscovery rates. Rapid and cheap genome sequencing as well as the advent of bioinformatical analysis tools for biosynthetic gene cluster identification in combination with mass spectrometry-based molecular networking facilitated the tedious process of dereplication. In recent years several studies have been dedicated to accessing the biosynthetic potential of Actinomyces species, especially streptomycetes, by using integrated genomic and metabolomic screening in order to boost the discovery rate of new antibiotics. This review aims to present the various possible applications of this approach as well as the newly discovered molecules, covering studies between 2014 and 2021. Finally, the effectiveness of this approach with regard to find new bioactive agents from actinomycetes will be evaluated.

Graphical abstract, Highlights • MALDI-TOF MS is applicable as high-resolution and high-throughput tool. • The classification and characterization of cultivable microorganisms is targeted. • Advantageous are its simple sample preparation and short measurement time. • It accelerates the dereplication of isolates from large-scale screening campaigns. • Applications for studying microbial diversity and future trends are discussed., Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) has become one of the most popular methods for the rapid, cost-effective and accurate classification and characterization of cultivable microorganisms. Due to its simple sample preparation and short measurement time, MALDI-TOF MS is an excellent choice for the high-throughput study of microbial isolates from rhizospheres or plants grown under diverse environmental conditions. While clinical isolates have a higher identification rate than environmental isolates due to the focus of commercial mass spectral libraries on the former, no identification is necessary in the dereplication step of large environmental studies. The grouping of large sets of isolates according to their intact protein profiles can be performed without knowledge of their taxonomy. Thus, this method is easily applicable to environmental samples containing microorganisms from yet undescribed phylogenetic origins. The main strategies applied to achieve effective dereplication are, first, expanding existing mass spectral libraries and, second, using an additional statistical analysis step to group measured mass spectra and identify unique isolates. In this review, these aspects are addressed. It closes with a prospective view on how MALDI-TOF MS-based microbial characterisation can accelerate the exploitation of plant-associated microbiota.

Made available in DSpace on 2019-10-04T12:37:45Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-03-01. Added 1 bitstream(s) on 2019-10-09T18:34:07Z : No. of bitstreams: 1 S0102-695X2019000200191.pdf: 1411392 bytes, checksum: 5a8ec7aa769f17312d1bd6c4d5ed4f32 (MD5) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) EU Finatec Natural products have been the most valuable source of chemical compounds in the discovery of novel medicines. Secondary metabolites from terrestrial and marine organisms have found considerable use in the treatment of numerous diseases and have been considered lead molecules both in their natural form and as templates for medicinal chemistry. Brazil has an exceptionally rich biodiversity, and a valuable source of secondary metabolites that can be useful for the development of bioproducts. Ipomoea species, Convolvulaceae, are mostly found in tropical and sub-tropical regions, including South America and many are used for nutritional and medicinal purposes. Ipomoea procumbens Mart. & Choisy is endemic from South America, and this is the first study reported on the chemical composition and biological activities of this species. The present work reports the tentatively identification of natural products present in the extracts using a high performance liquid chromatography-high resolution mass spectrometry method. Additionally, the antioxidant and antifungal biological activities of the leaves, roots and steams extracts and fractions of this species were evaluated. While for the antioxidant activity the hydromethanol fractions (leaves, stem and roots) were more active, the methanol fractions of leaves and stem provided better results for the antifungal assay. (C) 2018 Published by Elsevier Editora Ltda. on behalf of Sociedade Brasileira de Farmacognosia. Univ Paris 05, Lab Pharmacognosy, UMR CNRS Ctr Natl Rech Sci, Fac Pharm, Paris, France Univ Estadual Paulista, Inst Quim, Nucleo Bioensaios Biossintese & Ecofisiol Prod Na, Dept Quim Organ, Araraquara, SP, Brazil Univ Estadual Londrina, Dept Quim, Londrina, PR, Brazil Univ Estadual Paulista, Fac Ciencias Farmaceut, Dept Anal Clin, Lab Micol Clin, Araraquara, SP, Brazil Secretaria Meio Ambiente Estado Sao Paulo, Inst Bot, Sao Paulo, SP, Brazil Univ Estadual Paulista, Inst Quim, Nucleo Bioensaios Biossintese & Ecofisiol Prod Na, Dept Quim Organ, Araraquara, SP, Brazil Univ Estadual Paulista, Fac Ciencias Farmaceut, Dept Anal Clin, Lab Micol Clin, Araraquara, SP, Brazil FAPESP: 2010/52327-5 FAPESP: 2013/07600-3 EU: 269301 FAPESP: 2010/17329-7 Finatec: 120/2017

Microorganisms associated with termites are an original resource for identifying new chemical scaffolds or active metabolites. A molecular network was generated from a collection of strain extracts analyzed by liquid chromatography coupled to tandem high-resolution mass spectrometry, a molecular network was generated, and activities against the human pathogens methicillin-resistant Staphylococcus aureus, Candida albicans and Trichophyton rubrum were mapped, leading to the selection of a single active extract of Penicillium sclerotiorum SNB-CN111. This fungal species is known to produce azaphilones, a colorful family of polyketides with a wide range of biological activities and economic interests in the food industry. By exploring the molecular network data, it was shown that the chemical diversity related to the P. sclerotiorum metabolome largely exceeded the data already reported in the literature. According to the described fragmentation pathways of protonated azaphilones, the annotation of 74 azaphilones was proposed, including 49 never isolated or synthesized thus far. Our hypothesis was validated by the isolation and characterization of eight azaphilones, among which three new azaphilones were chlorogeumasnol (63), peniazaphilone E (74) and 7-deacetylisochromophilone VI (80).

The discovery and identification of novel natural products of medicinal importance in the herbal medicine industry becomes a challenge. The complexity of this process can be reduced by dereplication strategies. The current study includes a method based on high-performance liquid chromatography (HPLC), using the evaporative light scattering detector (ELSD) to identify the 12 most common secondary metabolites in plant extracts. Twelve compounds including rutin, taxifolin, quercetin, apigenin, kaempferol, betulinic acid, oleanolic acid, betulin, lupeol, stigmasterol, and β-sitosterol were analyzed simultaneously. The polarity of the compounds varied greatly from highly polar (flavonoids) to non-polar (triterpenes and sterols). This method was also tested for HPLC-DAD and HPLC-ESI-MS/MS analysis. Oleanolic acid and ursolic acid could not be separated in HPLC-ELSD analysis but were differentiated using LC-ESI-MS/MS analysis due to different fragment ions. The regression values (R2 > 0.996) showed good linearity in the range of 50–1000 µg/mL for all compounds. The range of LOD and LOQ values were 7.76–38.30 µg/mL and 23.52–116.06 µg/mL, respectively. %RSD and % trueness values of inter and intraday studies were mostly

Solid-phase extraction embedded dialysis (SPEED technology) is an innovative procedure developed to physically separate in-situ, during the cultivation, the mycelium of filament forming microorganisms, such as actinomycetes and fungi, and the XAD-16 resin used to trap the secreted specialized metabolites. SPEED consists of an external nylon cloth and an internal dialysis tube containing the XAD resin. The dialysis barrier selects the molecular weight of the trapped compounds, and prevents the aggregation of biomass or macromolecules on the XAD beads. The external nylon promotes the formation of a microbial biofilm, making SPEED a biofilm supported cultivation process. SPEED technology was applied to the marine Streptomyces albidoflavus 19-S21, isolated from a core of a submerged Kopara sampled at 20 m from the border of a saltwater pond. The chemical space of this strain was investigated effectively using a dereplication strategy based on molecular networking and in-depth chemical analysis. The results highlight the impact of culture support on the molecular profile of Streptomyces albidoflavus 19-S21 secondary metabolites.

Microorganisms associated with termites are an original resource for identifying new chemical scaffolds or active metabolites. A molecular network was generated from a collection of strain extracts analyzed by liquid chromatography coupled to tandem high-resolution mass spectrometry, a molecular network was generated, and activities against the human pathogens methicillin-resistant Staphylococcus aureus, Candida albicans and Trichophyton rubrum were mapped, leading to the selection of a single active extract of Penicillium sclerotiorum SNB-CN111. This fungal species is known to produce azaphilones, a colorful family of polyketides with a wide range of biological activities and economic interests in the food industry. By exploring the molecular network data, it was shown that the chemical diversity related to the P. sclerotiorum metabolome largely exceeded the data already reported in the literature. According to the described fragmentation pathways of protonated azaphilones, the annotation of 74 azaphilones was proposed, including 49 never isolated or synthesized thus far. Our hypothesis was validated by the isolation and characterization of eight azaphilones, among which three new azaphilones were chlorogeumasnol (63), peniazaphilone E (74) and 7-deacetylisochromophilone VI (80).

International audience; The recent revival of the study of organic natural products as renewable sources of medicinal drugs, cosmetics, dyes, and materials motivated the creation of general purpose structural databases. Dereplication, the efficient identification of already reported compounds, relies on the grouping of structural, taxonomic and spectroscopic databases that focus on a particular taxon (species, genus, family, order, etc.). A set of freely available python scripts, CNMR_Predict, is proposed for the quick supplementation of taxon oriented search results from the naturaL prOducTs occUrrences database (LOTUS, lotus.naturalproducts.net) with predicted carbon-13 nuclear magnetic resonance data from the ACD/Labs CNMR predictor and DB software (acdlabs.com) to provide easily searchable databases. The database construction process is illustrated using Brassica rapa as a taxon example

International audience; Chamaecrista diphylla (L.) Greene (Fabaceae/Caesalpiniaceae) is a herbaceous plant that is widely distributed throughout the Americas. Plants from this genus have been used in traditional medicine as a laxative, to heal wounds, and to treat ulcers, snake and scorpion bites. In the present study, we investigated the chemical composition of Chamaecrista diphylla leaves through a mass spectrometry molecular network approach. The oxygen radical absorbance capacity (ORAC) for the ethanolic extract, enriched fractions and isolated compounds was assessed. Overall, thirty-five compounds were annotated for the first time in C. diphylla. Thirty-two of them were reported for the first time in the genus. The isolated compounds 9, 12, 24 and 33 showed an excellent antioxidant capacity, superior to the extract and enriched fractions. Bond dissociation energy calculations were performed to explain and sustain the antioxidant capacity found. According to our results, the leaves of C. diphylla represent a promising source of potent antioxidant compounds.

Chamaecrista diphylla (L.) Greene (Fabaceae/Caesalpiniaceae) is a herbaceous plant that is widely distributed throughout the Americas. Plants from this genus have been used in traditional medicine as a laxative, to heal wounds, and to treat ulcers, snake and scorpion bites. In the present study, we investigated the chemical composition of Chamaecrista diphylla leaves through a mass spectrometry molecular network approach. The oxygen radical absorbance capacity (ORAC) for the ethanolic extract, enriched fractions and isolated compounds was assessed. Overall, thirty-five compounds were annotated for the first time in C. diphylla. Thirty-two of them were reported for the first time in the genus. The isolated compounds 9, 12, 24 and 33 showed an excellent antioxidant capacity, superior to the extract and enriched fractions. Bond dissociation energy calculations were performed to explain and sustain the antioxidant capacity found. According to our results, the leaves of C. diphylla represent a promising source of potent antioxidant compounds.

TQMD is a tool which downloads, stores and produces lists of dereplicated prokaryotic genomes. It has been developed to counter the ever-growing number of prokaryotic genomes and their uneven taxonomic distribution. It is based on word-based alignment-free methods (k-mers), an iterative single-linkage approach and a divide-and-conquer strategy to remain both efficient and scalable. We studied the performance of TQMD by verifying the influence of its parameters and heuristics on the clustering outcome. We further compared TQMD to two other dereplication tools (dRep and Assembly-Dereplicator). Our results showed that TQMD is optimized to dereplicate at high taxonomic levels (phylum/class), whereas the other dereplication tools are optimized for lower taxonomic levels (species/strain), making TQMD complementary to the existing dereplicating tools. TQMD is available at .

Chamaecrista diphylla (L.) Greene (Fabaceae/Caesalpiniaceae) is a herbaceous plant that is widely distributed throughout the Americas. Plants from this genus have been used in traditional medicine as a laxative, to heal wounds, and to treat ulcers, snake and scorpion bites. In the present study, we investigated the chemical composition of Chamaecrista diphylla leaves through a mass spectrometry molecular network approach. The oxygen radical absorbance capacity (ORAC) for the ethanolic extract, enriched fractions and isolated compounds was assessed. Overall, thirty-five compounds were annotated for the first time in C. diphylla. Thirty-two of them were reported for the first time in the genus. The isolated compounds 9, 12, 24 and 33 showed an excellent antioxidant capacity, superior to the extract and enriched fractions. Bond dissociation energy calculations were performed to explain and sustain the antioxidant capacity found. According to our results, the leaves of C. diphylla represent a promising source of potent antioxidant compounds.

Three species of the lichen Usnea (U. baileyi (Stirt.) Zahlbr., U. bismolliuscula Zahlbr. and U. pectinata Stirt.) and nine associated endolichenic fungi (ELF) were evaluated using a metabolomics approach. All investigated lichen crude extracts afforded antibacterial activity against Staphylococcus aureus (minimum inhibitory concentration (MIC): 0.0625 mg/mL), but none was observed against Escherichia coli, while the ELF extract Xylaria venustula was found to be the most active against S. aureus (MIC: 2.5 mg/mL) and E. coli (MIC: 5 mg/mL). X. venustula was fractionated and tested for to determine its antibacterial activity. Fractions XvFr1 to 5 displayed bioactivities against both test bacteria. Selected crude extracts and fractions were subjected to metabolomics analyses using high-resolution LC–MS. Multivariate analyses showed the presence of five secondary metabolites unique to bioactive fractions XvFr1 to 3, which were identified as responsible for the antibacterial activity of X. venustula. The p-values of these metabolites were at the margin of significance level, with methyl xylariate C (P_60) being the most significant. However, their high variable importance of projection (VIP) scores (&gt, 5) suggest these metabolites are potential diagnostic metabolites for X. venustula for “dual” bioactivity against S. aureus and E. coli. The statistical models also showed the distinctiveness of metabolites produced by lichens and ELF, thus supporting our hypotheses of ELF functionality similar to plant endophytes.

International audience; While depsidones, depsides or dibenzofuran-like compounds dominate the chemical composition of lichens, the cyanolichen Nephroma laevigatum affords a diversity of quinoid pigments represented by chlorinated anthraquinones derived from emodin and new bianthrones resulting from the homo- or heterodimerization of monomers. Bianthrones were pointed out from the dichloromethane extract by MS/MS-based molecular networking, then isolated and characterized on the basis of extensive spectroscopic analyzes and GIAO NMR shift calculation followed by CP3 analyzes.

Stryphnodendron pulcherrimum is a species known to have a high content of tannins. Accordingly, its preparations are used in southern Pará, Brazil, for their anti-inflammatory and antimicrobial activities, but so far, its chemical profile composition remains essentially unknown. We herein describe the compounds present in a hydro-acetonic extract from S. pulcherrimum leaves as revealed by dereplication via ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry. The data were combined with spectral organization, spectral matching through the Global Natural Products Social platform, in silico annotation and taxonomical ponderation. Several types of phenolic compounds were identified such as gallic acids, flavan-3-ols and flavone-like compounds. From these, 5 have been recently reported by our group, whereas 44 are reported here for the first time in this tree species, and 41 (out of 49) for this genus. The results highlight the possible role of Stryphnodendron pulcherrimum as a renewable source for natural bioactive products with potential pharmaceutical applications.

International audience; Bacterial genome sequencing has revealed a vast number of novel biosynthetic gene clusters (BGC) with potential to produce bioactive natural products. However, the biosynthesis of secondary metabolites by bacteria is often silenced under laboratory conditions, limiting the controlled expression of natural products. Here we describe an integrated methodology for the construction and screening of an elicited and pre-fractionated library of marine bacteria. In this pilot study, chemical elicitors were evaluated to mimic the natural environment and to induce the expression of cryptic BGCs in deep-sea bacteria. By integrating high-resolution untargeted metabolomics with cheminformatics analyses, it was possible to visualize, mine, identify and map the chemical and biological space of the elicited bacterial metabolites. The results show that elicited bacterial metabolites correspond to ~45% of the compounds produced under laboratory conditions. In addition, the elicited chemical space is novel (~70% of the elicited compounds) or concentrated in the chemical space of drugs. Fractionation of the crude extracts further evidenced minor compounds (~90% of the collection) and the detection of biological activity. This pilot work pinpoints strategies for constructing and evaluating chemically diverse bacterial natural product libraries towards the identification of novel bacterial metabolites in natural product-based drug discovery pipelines.

Many natural extracts used as cosmetic ingredients are available as solutions prepared in high-boiling-point solvents, called carrier solvents, such as propylene glycol (1,2-propanediol), propanediol (1,3-propanediol) and glycerin. The upstream chemical profiling of these extracts represents a major asset for the cosmetic industry, because it accelerates product development. A new workflow for the rapid characterization of the main metabolites present in natural extracts diluted in propylene glycol and 1,3-propanediol is presented here as an extension of previous works on glycerin-containing extracts. This method is an optimized version of a well-established dereplication procedure and consists of a fractionation by centrifugal partition chromatography followed by 13C nuclear magnetic resonance analysis and dedicated data processing. The concentration by evaporation under reduced pressure was considered as a pertinent preliminary step, particularly adapted to the analysis of highly diluted extracts. A dried hydro-ethanolic extract of Leontopodium alpinum Cass. was prepared at laboratory scale and used for method validation. Three solutions at 5% wt. of dry extract were prepared with propylene glycol/water (1:1), 1,3-propanediol/water (1:1) and glycerin/water (1:1) as carrier solvents. The dereplication workflow was applied to the three resulting L. alpinum extracts. Each study led to the quick identification of 26 metabolites including five flavonoids (luteolin and its derivatives), five hydroxycinnamic acids (among which are leontopodic acids), sugars and organic acids.

Lichens are symbiotic fungi, the chemistry of which has been used by humans since antiquity. They were however not studied with modern tools in metabolomics, which contributed in cementing the idea that lichens have poor chemical diversity. They are traditionally associated with 1050 molecules, although this figure is dated and does not seem to match the number of lichen species (19 387). In the field of metabolomics, LC-MS and dereplication using databases are frequently used for chemical profiling. These databases are nevertheless not adapted to the study of lichens, since they produce unique metabolites. Thus, several specific databases were produced herein, using the literature and pure standards analysed by LC-MS. Additional tools were developed to better the dereplication efficiency by predicting the molecules from the ions detected. These were all applied to the analysis of 300 lichen samples to highlight the chemical diversity of lichens using modern techniques. This resulted in the prediction of 8000 molecules with varying precision. The detailed investigation of the results has yet to be completed, but these already indicate that lichens are host to an underestimated chemical diversity that remains to be explored., Les lichens sont des champignons symbiotiques dont la chimie est exploitée par l’Homme depuis l’antiquité. Ils n’ont cependant pas été intégrés aux études de métabolomique récentes ce qui a installé l’idée que les lichens sont pauvres en molécules. 1050 molécules leur sont classiquement attribuées, bien que ce décompte date et qu’il semble éloigné de ce qui pourrait être attendu pour un mode de vie concernant 19 387 espèces. En métabolomique, LC-MS et la déréplication à l’aide de bases de données sont régulièrement usitées pour permettre le profilage des échantillons. Ces bases de données ne sont cependant pas adaptées à l’étude des lichens, qui produisent principalement des molécules qui leur sont uniques. Dans cette optique, plusieurs bases de données spécifiques aux lichens ont été créées ici, en utilisant des données de la littérature ainsi qu’en produisant des données spectrales. Des outils ont été créées pour améliorer la déréplication par la prédiction des molécules contenues dans les extraits à partir des ions qu’elles produisent. Tout ceci a été appliqué à l’analyse de 300 échantillons de lichens pour mettre en évidence la diversité chimique de ces champignons à l’aide de techniques modernes. Ceci a permis de prédire quelque 8000 molécules avec des degrés de certitude variables. L’étude détaillée des résultats pour mettre à jour les connaissances sur les lichens reste à faire, mais ceux-ci permettent déjà d’avancer que ces organismes sont à l’origine d’une chimie sous-estimée et qui reste encore à explorer.

A major challenge for LC-MS analysis is the ability to compare data between laboratories and across instrument platforms. Currently, mycotoxin determination relies on dereplication strategies based on physicochemical properties such as the m/z of the precursor and product ions. Unlike these intrinsic properties, retention time (tR) is an extrinsic property impacted by LC conditions, including mobile phases and column chemistry, making exchange of data between groups difficult. To address this, we are introducing the concept of incorporating an electrospray compatible, retention index (RI) system based on a series of N-alkylpyridinium-3-sulfonates (NAPS) into routine mycotoxin determination. These standards of differing alkyl chain length span RI units from 100 to 2000, are UV active and have fixed positive and negative charges for electrospray ionization in either mode. Using high resolution LC-MS/MS, the RIs of 96 mycotoxins and fungal secondary metabolites were normalized as a proof of concept with the NAPS RI system under multiple pH, column and gradient chromatographic conditions. This method was then applied to the analysis of a crude extract of Penicillium roqueforti, where we were able to decrease the number of false positives by implementing an RI filter as well as a secondary correction factor. Additionally, we developed software that allows users to convert published RIs to a predicted tR values. Integration of the NAPS RI system into routine LC-MS analysis will improve compound identifications and help facilitate ease of data sharing.

International audience; Natural products are a reliable source of bioactive molecules and represent an industrial and pharmaceu-tical stake. Indeed, the model yeast species Saccharomyces cerevisiae is a well-known eukaryotic organismlargely used as a biotechnological tool, but still a topical subject of study. In this work, the exploration ofSaccharomyces cerevisiae is taken further through an untargeted metabolomics workflow. The aim is toenrich databases and bring new information about the standard S. cerevisiae strain in a given medium.Analytical methods and bioinformatics tools were combined in a high-throughput methodology useableto dereplicate many types of biological extracts and cartography secondary metabolites. Ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analyses were carriedout and spectral data were pre-processed to build molecular networks. Annotations were attributedto compounds through comparison with databases and manual investigation of networks. Ultra-high-resolution Fourier-transform ion cyclotron resonance mass spectrometry (FTICR-MS) brought additionalinformation thanks to a higher dynamic range and enhanced UHPLC-MS/MS results by unveiling ambi-guities and bringing accurate molecular formulae. Therefore, accurate and reliable annotated featuresresulted from the UHPLC-MS/MS data while FTICR-MS provided an overall cartography of metabolitesthanks to van Krevelen diagrams. Various small molecules such as amino acids derivatives and indolealkaloids have been determined for the first time in this yeast. The complementarity of FTICR-MS and UHPLC-MS/MS for secondary metabolite annotation brought this new mapping of S. cerevisiae.

MOLECULAR NETWORKS: AN ANALYSIS ON ANNOTATIONS AND DISCOVERY OF NEW ASSETS. To speed up the discovery of bioactive natural products (NP), chemists have sought advanced approaches in analytical and computational chemistry in attempt to organize and extract information from large data sets. In this sense, the molecular networks (MN) successfully organized enormous sets of mass spectrometry (MS) data together with samples metadata information in an intuitive visualization in the spectral similarity networks format. GNPS (Global Natural Products Social Molecular Networking), a free online platform for storing and processing MSn data, is a leading application of spectral matching with public databases aimed at the dereplication and discovery of new bioactive products through molecular networks. In this review, we address the concept of GNPS spectral similarity networks, as well as their complementary computational tools, benefits and limitations applied in NP studies associated with dereplication, chemical ecology, functional genetics and determination of biosynthetic pathways.

International audience; This study presents the bio-guided chemical investigation of 80% methanol extract of aerial parts of bladder tree or Colutea arborescens. Liquid-liquid partitioning in solvents of increasing polarity combined with biological screening showed that the EtOAc and n-BuOH soluble fractions were the most active parts of the extract. These fractions were chemically profiled by a 13 C NMR-based dereplication method, resulting in the identification of fifteen compounds. The dereplication process was completed by purification of minor compounds of the n-BuOH fraction. Fourteen known compounds (1-14) were isolated, including two tri-glycosylated flavonoids containing an apiofuranose unit (12 and 14). Their structures were elucidated by spectroscopic methods including 1D and 2D NMR and high-resolution electrospray ionization mass spectrometry. The antioxidant activity of fractions and isolated compounds were evaluated using 2,2,1-diphenyl-1-picrylhydrazyl and hydroxyl radicals scavenging and by cupric ion reducing antioxidant capacity assays. In parallel, their inhibitory properties against mushroom tyrosinase and human neutrophil elastase enzymes were assessed. Quercetin-type flavonoids showed the best antioxidant activities and a tri-glycosylated quercetin exhibited both antioxidant and anti-elastase activities. Phenylethyl-glucoside exhibited a significant tyrosinase inhibitory activity.

open access article The role and importance of the identification of natural products are first discussed in the perspective of the study of secondary metabolites. The rapid identification of already reported compounds, or structural dereplication, is recognized as a key element in natural product chemistry. The biological taxonomy of metabolite producing organisms, the knowledge of metabolite molecular structures, and the availability of metabolite spectroscopic signatures are considered as the three pillars of structural dereplication. The role and the construction of databases is illustrated by references to the KNApSAcK, UNPD, CSEARCH, and COCONUT databases, and by the importance of calculated taxonomic and spectroscopic data as substitutes for missing or lost original ones. Two NMR-based tools, the PNMRP database that derives from UNPD, and KnapsackSearch, a database generator that provides taxonomically focused libraries of compounds, are proposed to the community of natural product chemists. The study of the alkaloids from Urceolina peruviana, a plant from the Andes used in traditional medicine for antibacterial and anticancer actions, has given the opportunity to test different approaches to dereplication, favoring the use of publicly available data sources.

The marine-derived fungus Stilbella fimetaria is a chemically talented fungus producing several classes of bioactive metabolites, including meroterpenoids of the ascochlorin family. The targeted dereplication of fungal extracts by UHPLC-DAD-QTOF-MS revealed the presence of several new along with multiple known ascochlorin analogues (19&ndash, 22). Their structures and relative configuration were characterized by 1D and 2D NMR. Further targeted dereplication based on a novel 1,4-benzoquinone sesquiterpene derivative, fimetarin A (22), resulted in the identification of three additional fimetarin analogues, fimetarins B&ndash, D (23&ndash, 25), with their tentative structures proposed from detailed MS/HRMS analysis. In total, four new and eight known ascochlorin/fimetarin analogues were tested for their antimicrobial activity, identifying the analogues with a 5-chloroorcylaldehyde moiety to be more active than the benzoquinone analogue. Additionally, the presence of two conjugated double bonds at C-2&prime, /C-3&prime, and C-4&prime, /C-5&prime, were found to be essential for the observed antifungal activity, whereas the single, untailored bonds at C-4&prime, and C-8&prime, /C-9&prime, were suggested to be necessary for the observed antibacterial activity.

Recovering and annotating bacterial genomes from metagenomes involves a series of complex computational tools that are often difficult to use for researches without a specialistic bioinformatic background. In this chapter we review all the steps that lead from raw reads to a collection of quality-controlled, functionally annotated bacterial genomes and propose a working protocol using state-of-the-art, open source software tools.

Made available in DSpace on 2021-06-25T10:26:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-01-01 This work has as the main focus, to analyze the behavior of physic-chemical variations from the fungus Xylaria sp., through the OSMAC (One Strain, Many Compounds) approach as an efficient way of obtaining new compounds. To perform such inductions and to explore the variability of the metabolic network of this microorganism, a factorial design was designed to induce variability (or enhancement) of metabolites. In view of chemometric insights, the planned inductions were imposed on the microorganism and variations in the chemical profile were observed in the crude extracts. Through mass spectrometry (HR-ESI–MS) and nuclear magnetic resonance-based profiles, combined with multivariate analysis through Principal Component Analysis (PCA), it was observed a marked variability of signals, confirming the efficacy in the metabolic alteration, defining the culture medium and agitation as the most important variables in the metabolic variability of the fungus. However, the extract mass is more significant for the agitation variable, and there is no relationship between the mass of crude extract and the amount of molecular signals of the complex matrices studied. Instituto Federal de Educação Ciência e Tecnologia de Rondônia IFRO, Campus Ji-Paraná, Street Rio Amazonas, 151 Instituto Federal de Educação Ciência e Tecnologia de Rondônia IFRO University of Manchester UoM Universidade Federal de São Carlos UFSCar Universidade Estadual Paulista “Júlio de Mesquita Filho” UNESP Universidade Estadual Paulista “Júlio de Mesquita Filho” UNESP

Made available in DSpace on 2022-04-29T08:36:49Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-01-01 To speed up the discovery of bioactive natural products (NP), chemists have sought advanced approaches in analytical and computational chemistry in attempt to organize and extract information from large data sets. In this sense, the molecular networks (MN) successfully organized enormous sets of mass spectrometry (MS) data together with samples metadata information in an intuitive visualization in the spectral similarity networks format. GNPS (Global Natural Products Social Molecular Networking), a free online platform for storing and processing MSn data, is a leading application of spectral matching with public databases aimed at the dereplication and discovery of new bioactive products through molecular networks. In this review, we address the concept of GNPS spectral similarity networks, as well as their complementary computational tools, benefits and limitations applied in NP studies associated with dereplication, chemical ecology, functional genetics and determination of biosynthetic pathways. Departamento de Química Orgânica Instituto de Química Universidade Estadual Paulista, SP Departamento de Ciências Biomoleculares Faculdade de Ciências Farmacêuticas Universidade de São Paulo, SP Instituto Multidisciplinar em Saúde Campus Anísio Teixeira Universidade Federal da Bahia, BA Instituto de Química de São Carlos Universidade de São Paulo, SP Universidade de Brasília Campus Universitário Darcy Ribeiro, DF Departamento de Química Orgânica Instituto de Química Universidade Estadual Paulista, SP

Simple Summary In this study, we determined the bioactivities and chemical natures of three species of lichen Usnea and their associated endolichenic fungi (ELF) through metabolomics. We found significant differences in the antibacterial activities and the metabolites produced by the host lichen and its ELF, with the latter targeting a wider scope of organisms. We also discovered potential key metabolites produced by ELF that are yet to be reported. This study shows the application of metabolomics in rapidly identifying bioactive metabolites that are of significance in the discovery of new drugs. Abstract Three species of the lichen Usnea (U. baileyi (Stirt.) Zahlbr., U. bismolliuscula Zahlbr. and U. pectinata Stirt.) and nine associated endolichenic fungi (ELF) were evaluated using a metabolomics approach. All investigated lichen crude extracts afforded antibacterial activity against Staphylococcus aureus (minimum inhibitory concentration (MIC): 0.0625 mg/mL), but none was observed against Escherichia coli, while the ELF extract Xylaria venustula was found to be the most active against S. aureus (MIC: 2.5 mg/mL) and E. coli (MIC: 5 mg/mL). X. venustula was fractionated and tested for to determine its antibacterial activity. Fractions XvFr1 to 5 displayed bioactivities against both test bacteria. Selected crude extracts and fractions were subjected to metabolomics analyses using high-resolution LC–MS. Multivariate analyses showed the presence of five secondary metabolites unique to bioactive fractions XvFr1 to 3, which were identified as responsible for the antibacterial activity of X. venustula. The p-values of these metabolites were at the margin of significance level, with methyl xylariate C (P_60) being the most significant. However, their high variable importance of projection (VIP) scores (>5) suggest these metabolites are potential diagnostic metabolites for X. venustula for “dual” bioactivity against S. aureus and E. coli. The statistical models also showed the distinctiveness of metabolites produced by lichens and ELF, thus supporting our hypotheses of ELF functionality similar to plant endophytes.

The discovery of novel natural products (NPs) that will serve as lead structures has to be an ongoing effort to fill the respective development pipelines. However, identification of NPs, which possess a potential for application in e.g., the pharma or agro sector, must be as cost effective and fast as possible. Furthermore, the amount of sample available for initial testing is usually very limited, not least because of the fact that the impact on the environment, i.e., the sampled biosystem, should be kept minimal. Here, our pipeline SeaPEPR is described, in which a primary bioactivity screening of crude extracts is combined with the analysis of their metabolic fingerprint. This enabled prioritization of samples for subsequent microfractionation and dereplication of the active compounds early in the workflow. As a case study, 76 marine sponge-derived extracts were screened against a microbial screening panel. Thereunder, human pathogenic bacteria (Escherichia coli ATCC35218 and Staphylococcus aureus ATCC33592) and yeast (Candida albicans FH2173), as well as the phytopathogenic fungus Septoria tritici MUCL45407. Overall, nine extracts revealed activity against at least one test organism. Metabolic fingerprinting enabled assigning four active extracts into one metabolic group, therefore, one representative was selected for subsequent microfractionation. Dereplication of the active fractions showed a new dibrominated aplysinopsin and a hypothetical chromazonarol stereoisomer derivative. Furthermore, inhibitory activity against the common plant pest Septoria tritici was discovered for NPs of marine origin.