Evaluation of Data-Dependent MS/MS Acquisition Parameters for Non-Targeted Metabolomics and Molecular Networking of Environmental Samples: Focus on the Q Exactive Platform.

Non-targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a widely used tool for metabolomics analysis, enabling the detection and annotation of small molecules in complex environmental samples. Data-dependent acquisition (DDA) of product ion spectra is thereby currently one of the most frequently applied data acquisition strategies. The optimization of DDA parameters is central to ensuring high spectral quality, coverage, and number of compound annotations. Here, we evaluated the influence of 10 central DDA settings of the Q Exactive mass spectrometer on natural organic matter samples from ocean, river, and soil environments. After data analysis with classical and feature-based molecular networking using MZmine and GNPS, we compared the total number of network nodes, multivariate clustering, and spectrum quality-related metrics such as annotation and singleton rates, MS/MS placement, and coverage. Our results show that automatic gain control, microscans, mass resolving power, and dynamic exclusion are the most critical parameters, whereas collision energy, TopN, and isolation width had moderate and apex trigger, monoisotopic selection, and isotopic exclusion minor effects. The insights into the data acquisition ergonomics of the Q Exactive platform presented here can guide new users and provide them with initial method parameters, some of which may also be transferable to other sample types and MS platforms.

The Cyanobacterial "Nutraceutical" Phycocyanobilin Inhibits Cysteine Protease Legumain.

The blue biliprotein phycocyanin, produced by photo-autotrophic cyanobacteria including spirulina (Arthrospira) and marketed as a natural food supplement or "nutraceutical," is reported to have anti-inflammatory, antioxidant, immunomodulatory, and anticancer activity. These diverse biological activities have been specifically attributed to the phycocyanin chromophore, phycocyanobilin (PCB). However, the mechanism of action of PCB and the molecular targets responsible for the beneficial properties of PCB are not well understood. We have developed a procedure to rapidly cleave the PCB pigment from phycocyanin by ethanolysis and then characterized it as an electrophilic natural product that interacts covalently with thiol nucleophiles but lacks any appreciable cytotoxicity or antibacterial activity against common pathogens and gut microbes. We then designed alkyne-bearing PCB probes for use in chemical proteomics target deconvolution studies. Target identification and validation revealed the cysteine protease legumain (also known as asparaginyl endopeptidase, AEP) to be a target of PCB. Inhibition of this target may account for PCB's diverse reported biological activities.

Biosynthesis and antifungal activity of fungus-induced O-methylated flavonoids in maize.

Fungal infection of grasses, including rice (Oryza sativa), sorghum (Sorghum bicolor), and barley (Hordeum vulgare), induces the formation and accumulation of flavonoid phytoalexins. In maize (Zea mays), however, investigators have emphasized benzoxazinoid and terpenoid phytoalexins, and comparatively little is known about flavonoid induction in response to pathogens. Here, we examined fungus-elicited flavonoid metabolism in maize and identified key biosynthetic enzymes involved in the formation of O-methylflavonoids. The predominant end products were identified as two tautomers of a 2-hydroxynaringenin-derived compound termed xilonenin, which significantly inhibited the growth of two maize pathogens, Fusarium graminearum and Fusarium verticillioides. Among the biosynthetic enzymes identified were two O-methyltransferases (OMTs), flavonoid OMT 2 (FOMT2), and FOMT4, which demonstrated distinct regiospecificity on a broad spectrum of flavonoid classes. In addition, a cytochrome P450 monooxygenase (CYP) in the CYP93G subfamily was found to serve as a flavanone 2-hydroxylase providing the substrate for FOMT2-catalyzed formation of xilonenin. In summary, maize produces a diverse blend of O-methylflavonoids with antifungal activity upon attack by a broad range of fungi.

Specialized Metabolite-Mediated Predation Defense in the Marine Actinobacterium Salinispora.

The obligate marine actinobacterial genus Salinispora has become a model organism for natural product discovery, yet little is known about the ecological functions of the compounds produced by this taxon. The aims of this study were to assess the effects of live cultures and culture extracts from two Salinispora species on invertebrate predators. In choice-based feeding experiments using the bacterivorous nematode Caenorhabditis elegans, live cultures of both Salinispora species were less preferred than Escherichia coli. When given a choice between the two species, C. elegans preferred S. areniolca over S. tropica. Culture extracts from S. tropica deterred C. elegans, while those from S. arenicola did not, suggesting that compounds produced by S. tropica account for the feeding deterrence. Bioactivity-guided isolation linked compounds in the lomaiviticin series to the deterrent activity. Additional assays using the marine polychaete Ophryotrocha siberti and marine nematodes further support the deterrent activity of S. tropica against potential predators. These results provide evidence that Salinispora natural products function as a defense against predation and that the strategies of predation defense differ between closely related species. IMPORTANCE Bacteria inhabiting marine sediments are subject to predation by bacterivorous eukaryotes. Here, we test the hypothesis that sediment-derived bacteria in the genus Salinispora produce biologically active natural products that function as a defense against predation. The results reveal that cultures and culture extracts of S. tropica deter feeding by Caenorhabditis elegans and negatively affect the habitat preference of a marine annelid (Ophryotrocha siberti). These activities were linked to the lomaiviticins, a series of cytotoxic compounds produced by S. tropica. Microbial natural products that function as a defense against predation represent a poorly understood trait that can influence community structure in marine sediments.

Chemical labeling strategies for small molecule natural product detection and isolation.

Covering: Up to 2020.It is widely accepted that small molecule natural products (NPs) evolved to carry out a particular ecological function and that these finely-tuned molecules can sometimes be appropriated for the treatment of disease in humans. Unfortunately, for the natural products chemist, NPs did not evolve to possess favorable physicochemical properties needed for HPLC-MS analysis. The process known as derivatization, whereby an NP in a complex mixture is decorated with a nonnatural moiety using a derivatizing agent (DA), arose from this sad state of affairs. Here, NPs are freed from the limitations of natural functionality and endowed, usually with some degree of chemoselectivity, with additional structural features that make HPLC-MS analysis more informative. DAs that selectively label amines, carboxylic acids, alcohols, phenols, thiols, ketones, and aldehydes, terminal alkynes, electrophiles, conjugated alkenes, and isocyanides have been developed and will be discussed here in detail. Although usually employed for targeted metabolomics, chemical labeling strategies have been effectively applied to uncharacterized NP extracts and may play an increasing role in the detection and isolation of certain classes of NPs in the future.

Discovery, Biosynthesis and Stress-Related Accumulation of Dolabradiene-Derived Defenses in Maize.

Terpenoids are a major component of maize (Zea mays) chemical defenses that mediate responses to herbivores, pathogens, and other environmental challenges. Here, we describe the biosynthesis and elicited production of a class of maize diterpenoids, named dolabralexins. Dolabralexin biosynthesis involves the sequential activity of two diterpene synthases, ENT-COPALYL DIPHOSPHATE SYNTHASE (ZmAN2) and KAURENE SYNTHASE-LIKE4 (ZmKSL4). Together, ZmAN2 and ZmKSL4 form the diterpene hydrocarbon dolabradiene. In addition, we biochemically characterized a cytochrome P450 monooxygenase, ZmCYP71Z16, which catalyzes the oxygenation of dolabradiene to yield the epoxides 15,16-epoxydolabrene (epoxydolabrene) and 3ß-hydroxy-15,16-epoxydolabrene (epoxydolabranol). The absence of dolabradiene and epoxydolabranol in Zman2 mutants under elicited conditions confirmed the in vivo biosynthetic requirement of ZmAN2. Combined mass spectrometry and NMR experiments demonstrated that much of the epoxydolabranol is further converted into 3ß,15,16-trihydroxydolabrene (trihydroxydolabrene). Metabolite profiling of field-grown maize root tissues indicated that dolabralexin biosynthesis is widespread across common maize cultivars, with trihydroxydolabrene as the predominant diterpenoid. Oxidative stress induced dolabralexin accumulation and transcript expression of ZmAN2 and ZmKSL4 in root tissues, and metabolite and transcript accumulation were up-regulated in response to elicitation with the fungal pathogens Fusarium verticillioides and Fusarium graminearum Consistently, epoxydolabranol significantly inhibited the growth of both pathogens in vitro at 10 µg mL-1, while trihydroxydolabrene-mediated inhibition was specific to Fverticillioides These findings suggest that dolabralexins have defense-related roles in maize stress interactions and expand the known chemical space of diterpenoid defenses as genetic targets for understanding and ultimately improving maize resilience.

Progress toward the Total Synthesis of Lymphostins: Preparation of a Functionalized Tetrahydropyrrolo[4,3,2-de]quinoline and Unusual Oxidative Dimerization.

The lymphostins are a family of closely related pyrrolo[4,3,2-de]quinoline natural products produced by Streptomyces and Salinispora actinobacteria. Neolymphostin A was recently shown to strongly inhibit phosphoinositide 3-kinase (PI3K) and the mammalian target of rapamycin (mTOR) in a covalent manner via conjugation to a catalytic lysine residue in the ATP-binding pocket of the enzymes, making this metabolite the first reported covalent kinase inhibitor from a bacterium. A flexible and efficient synthetic route toward these alkaloids would allow for improvements in their solubility, stability, and selectivity and help to deliver a viable drug candidate. We have since established a short synthesis to methyl 8-bromo-1,3,4,5-tetrahydropyrrolo[4,3,2-de]quinoline-4-carboxylate via a conjugate addition/intramolecular Ullman reaction sequence. However, attempts to oxidize this intermediate to the pyrrolo[4,3,2-de]quinoline characteristic of the lymphostins resulted in formation of either a 2-oxo-1,2-dihydropyrrolo[4,3,2-de]quinoline or an unusual N,C-linked tetrahydropyrroloquinoline-pyrroloquinoline heterodimer. We expect that key modifications to the tetrahydropyrroloquinoline intermediate prior to oxidation should prevent these side reactions and pave the way for the completion of the synthesis.

Nature's Combinatorial Biosynthesis Produces Vatiamides A-F.

Hybrid type I PKS/NRPS biosynthetic pathways typically proceed in a collinear manner wherein one molecular building block is enzymatically incorporated in a sequence that corresponds to gene arrangement. In this work, genome mining combined with the use of a fluorogenic azide-based click probe led to the discovery and characterization of vatiamides A-F, three structurally diverse alkynylated lipopeptides, and their brominated analogues, from the cyanobacterium Moorea producens ASI16Jul14-2. These derive from a unique combinatorial non-collinear PKS/NRPS system encoded by a 90 kb gene cluster in which an upstream PKS cassette interacts with three separate cognate NRPS partners. This is facilitated by a series of promiscuous intermodule PKS-NRPS docking motifs possessing identical amino acid sequences. This interaction confers a new type of combinatorial capacity for creating molecular diversity in microbial systems.

The role of inter-species interactions in Salinispora specialized metabolism.

Bacterial genome sequences consistently contain many more biosynthetic gene clusters encoding specialized metabolites than predicted by the compounds discovered from the respective strains. One hypothesis invoked to explain the cryptic nature of these gene clusters is that standard laboratory conditions do not provide the environmental cues needed to trigger gene expression. A potential source of such cues is other members of the bacterial community, which are logical targets for competitive interactions. In this study, we examined the effects of such interactions on specialized metabolism in the marine actinomycete Salinispora tropica. The results show that antibiotic activities and the concentration of some small molecules increase in the presence of co-occurring bacterial strains relative to monocultures. Some increases in antibiotic activity could be linked to nutrient depletion by the competitor as opposed to the production of a chemical cue. Other increases were correlated with the production of specific compounds by S. tropica. In particular, one interaction with a Vibrio sp. consistently induced antibiotic activity and was associated with parent ions that were unique to this interaction, although the associated compound could not be identified. This study provides insight into the metabolomic complexities of bacterial interactions and baseline information for future genome mining efforts.

Ecological implications of hypoxia-triggered shifts in secondary metabolism.

Members of the actinomycete genus Streptomyces are non-motile, filamentous bacteria that are well-known for the production of biomedically relevant secondary metabolites. While considered obligate aerobes, little is known about how these bacteria respond to periods of reduced oxygen availability in their natural habitats, which include soils and ocean sediments. Here, we provide evidence that the marine streptomycete strain CNQ-525 can reduce MnO2 via a diffusible mechanism. We investigated the effects of hypoxia on secondary metabolite production and observed a shift away from the antibiotic napyradiomycin towards 8-amino-flaviolin, an intermediate in the napyradiomycin biosynthetic pathway. We purified 8-amino-flaviolin and demonstrated that it is reversibly redox-active (midpoint potential -474.5 mV), indicating that it has the potential to function as an endogenous extracellular electron shuttle. This study provides evidence that environmentally triggered changes in secondary metabolite production may provide clues to the ecological functions of specific compounds, and that Gram-positive bacteria considered to be obligate aerobes may play previously unrecognized roles in biogeochemical cycling through mechanisms that include extracellular electron shuttling.

Thiol-Based Probe for Electrophilic Natural Products Reveals That Most of the Ammosamides Are Artifacts.

To date, 16 members of the ammosamide family of natural products have been discovered, and except for ammosamide D each of these metabolites is characterized by an unusual chlorinated pyrrolo[4,3,2-de]quinoline skeleton. Several ammosamides have been shown to inhibit quinone reductase 2, a flavoenzyme responsible for quelling toxic oxidative species in cells or for killing cancer cells outright. Treatment of the extract from an ammosamide-producing culture (Streptomyces strain CNR-698) with a thiol-based reagent designed to label electrophilic natural products produced an ammosamide C-thiol adduct. This observation led us to hypothesize, and then demonstrate through experimentation, that all of the other ammosamides are derived from ammosamide C via nonenzymatic processes involving exposure to nucleophiles, air, and light. Like many established electrophilic natural products, reaction with the thiol probe suggests that ammosamide C is itself an electrophilic natural product. Although ammosamide C did not show substantial cytotoxicity against cancer cells, its activity against a marine Bacillus bacterial strain may reflect its ecological role.

Sioxanthin, a novel glycosylated carotenoid, reveals an unusual subclustered biosynthetic pathway.

Members of the marine actinomycete genus Salinispora constitutively produce a characteristic orange pigment during vegetative growth. Contrary to the understanding of widespread carotenoid biosynthesis pathways in bacteria, Salinispora carotenoid biosynthesis genes are not confined to a single cluster. Instead, bioinformatic and genetic investigations confirm that four regions of the Salinispora tropica CNB-440 genome, consisting of two gene clusters and two independent genes, contribute to the in vivo production of a single carotenoid. This compound, namely (2'S)-1'-(ß-D-glucopyranosyloxy)-3',4'-didehydro-1',2'-dihydro-φ,ψ-caroten-2'-ol, is novel and has been given the trivial name 'sioxanthin'. Sioxanthin is a C40 -carotenoid, glycosylated on one end of the molecule and containing an aryl moiety on the opposite end. Glycosylation is unusual among actinomycete carotenoids, and sioxanthin joins a rare group of carotenoids with polar and non-polar head groups. Gene sequence homology predicts that the sioxanthin biosynthetic pathway is present in all of the Salinispora as well as other members of the family Micromonosporaceae. Additionally, this study's investigations of clustering of carotenoid biosynthetic genes in heterotrophic bacteria show that a non-clustered genome arrangement is more common than previously suggested, with nearly half of the investigated genomes showing a non-clustered architecture.

Antibacterial Marinopyrroles and Pseudilins Act as Protonophores.

Elucidating the mechanism of action (MoA) of antibacterial natural products is crucial to evaluating their potential as novel antibiotics. Marinopyrroles, pentachloropseudilin, and pentabromopseudilin are densely halogenated, hybrid pyrrole-phenol natural products with potent activity against Gram-positive bacterial pathogens like Staphylococcus aureus. However, the exact way they exert this antibacterial activity has not been established. In this study, we explore their structure-activity relationship, determine their spatial location in bacterial cells, and investigate their MoA. We show that the natural products share a common MoA based on membrane depolarization and dissipation of the proton motive force (PMF) that is essential for cell viability. The compounds show potent protonophore activity but do not appear to destroy the integrity of the cytoplasmic membrane via the formation of larger pores or interfere with the stability of the peptidoglycan sacculus. Thus, our current model for the antibacterial MoA of marinopyrrole, pentachloropseudilin, and pentabromopseudilin stipulates that the acidic compounds insert into the membrane and transport protons inside the cell. This MoA may explain many of the deleterious biological effects in mammalian cells, plants, phytoplankton, viruses, and protozoans that have been reported for these compounds.

Structure Elucidation of Antibiotics.

To date, there are hundreds of characterized natural products with antibacterial activity against pathogenic bacteria, and several have become bonafide antibiotic drugs. The development of antibacterial natural products into antibiotic drugs, both in the past and in the future, hinges upon an accurate description of the exact chemical structure of the compound. Bolstered by some form of mass spectrometry (MS), nuclear magnetic resonance (NMR) spectroscopy is the primary technique for elucidating the chemical structure of organic molecules including natural products. By combining various one-dimensional (1D) and two-dimensional (2D) experiments, the connectivity between atoms is established and a complete "picture" of the molecule is thereby revealed.

Flavoenzyme-catalyzed atropo-selective N,C-bipyrrole homocoupling in marinopyrrole biosynthesis.

Axially chiral biaryl compounds are frequently encountered in nature where they exhibit diverse biological properties. Many are biphenols that have C-C or C-O linkages installed by cytochrome P450 oxygenases that control the regio- and stereoselectivity of the intermolecular coupling reaction. In contrast, bipyrrole-coupling enzymology has not been observed. Marinopyrroles, produced by a marine-derived streptomycete, are the first 1,3'-bipyrrole natural products. On the basis of marinopyrrole's unusual bipyrrole structure, we explored its atropo-selective biosynthesis in Streptomyces sp. CNQ-418 in order to elucidate the N,C-bipyrrole homocoupling enzymology. Through a series of genetic experiments involving the discovery and heterologous expression of marinopyrrole biosynthesis genes, we report that two flavin-dependent halogenases catalyze the unprecedented homocoupling reaction.

Discovery of a Cryptic Nitro Intermediate in the Biosynthesis of the 3-(trans-2'-Aminocyclopropyl)alanine Moiety of Belactosin A.

Belactosin A, a ß-lactone proteasome inhibitor, contains a unique 3-(trans-2'-aminocyclopropyl)alanine moiety. We recently identified the biosynthetic gene cluster of the belactosin series from Streptomyces sp. UCK14. To shed light on the formation of the aminocyclopropylalanine, we established a heterologous pathway expression, constructed a set of gene deletion mutants, and performed feeding studies for a chemical complementation that include the incorporation of stable isotope-labeled precursors. We thereby show that, in the biosynthesis of this building block, a cryptic nitrocyclopropylalanine intermediate is generated from l-lysine. The subsequent reduction of the N-oxygenated precursor to the corresponding amine is mediated by the molybdopterin-dependent enzyme BelN.

Antibacterial Synnepyrroles from Human-Associated Nocardiopsis sp. Show Protonophore Activity and Disrupt the Bacterial Cytoplasmic Membrane.

Actinobacteria have traditionally been an important source of bioactive natural products, although many genera remain poorly explored. Here, we report a group of distinctive pyrrole-containing natural products, named synnepyrroles, from Nocardiopsis synnemataformans. Detailed structural characterization by mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy combined with isotope-labeling experiments revealed their molecular structures and biosynthetic precursors acetate, propionate, aspartate, and (for branched analogues) valine. The biosynthetic data points toward an unusual pathway for pyrrole formation via condensation of aspartate with diverse fatty acids that give rise to a unique pyrrole-3,4-dicarboxylate core and variable linear or terminally branched alkyl side chains. In addition, the bioactivity and mode of action of synnepyrrole A were characterized in Bacillus subtilis. Orienting assessment of the phenotype of synnepyrrole A-treated bacteria by high-resolution microscopy suggested the cytoplasmic membrane as the target structure. Further characterization of the membrane effects demonstrated dissipation of the membrane potential and intracellular acidification indicative of protonophore activity. At slightly higher concentrations, synnepyrrole A compromised the barrier function of the cytoplasmic membrane, allowing the passage of otherwise membrane-impermeable dye molecules.

Native metabolomics identifies the rivulariapeptolide family of protease inhibitors.

The identity and biological activity of most metabolites still remain unknown. A bottleneck in the exploration of metabolite structures and pharmaceutical activities is the compound purification needed for bioactivity assignments and downstream structure elucidation. To enable bioactivity-focused compound identification from complex mixtures, we develop a scalable native metabolomics approach that integrates non-targeted liquid chromatography tandem mass spectrometry and detection of protein binding via native mass spectrometry. A native metabolomics screen for protease inhibitors from an environmental cyanobacteria community reveals 30 chymotrypsin-binding cyclodepsipeptides. Guided by the native metabolomics results, we select and purify five of these compounds for full structure elucidation via tandem mass spectrometry, chemical derivatization, and nuclear magnetic resonance spectroscopy as well as evaluation of their biological activities. These results identify rivulariapeptolides as a family of serine protease inhibitors with nanomolar potency, highlighting native metabolomics as a promising approach for drug discovery, chemical ecology, and chemical biology studies.

Pharmacological properties of the marine natural product marinopyrrole A against methicillin-resistant Staphylococcus aureus.

The ongoing spread of methicillin-resistant Staphylococcus aureus (MRSA) strains in hospital and community settings presents a great challenge to public health and illustrates the urgency of discovering new antibiotics. Marinopyrrole A is a member of a structurally novel class of compounds identified from a species of marine-derived streptomycetes with evidence of antistaphylococcal activity. We show that marinopyrrole A has potent concentration-dependent bactericidal activity against clinically relevant hospital- and community-acquired MRSA strains, a prolonged postantibiotic effect superior to that of the current first-line agents vancomycin and linezolid, and a favorable resistance profile. Marinopyrrole A showed limited toxicity to mammalian cell lines (at >20× MIC). However, its antibiotic activity against MRSA was effectively neutralized by 20% human serum. A variety of marinopyrrole analogs were isolated from culture or synthetically produced to try to overcome the inhibitory effect of serum. While many of these compounds retained potent bactericidal effect against MRSA, their activities were also inhibited by serum. Marinopyrrole A has significant affinity for plastic and may therefore have potential as a potent anti-MRSA agent in cutaneous, intracatheter, or antibiotic-lock applications.

Total synthesis of the ammosamides.

The ammosamides A-C are chlorinated pyrrolo[4,3,2-de]quinoline metabolites isolated from the marine-derived Streptomyces strain CNR-698. The natural products, which possess a dense array of heteroatoms, were synthesized in 17-19 steps from 4-chloroisatin. That the five nitrogen atoms were introduced at the appropriate time and in a suitable oxidation state was key to the success of the total synthesis. Compared to synthetic deschloro ammosamide B, natural ammosamide B is much less susceptible to oxidative degradation.