Spatialized Metabolomic Annotation Combining MALDI Imaging and Molecular Networks.

MALDI mass spectrometry imaging has gained major interest in the field of chemical imaging. This technique makes it possible to locate tens to hundreds of ionic signals on the sample surface without any a priori. One of the current challenges is still the limited ability to annotate signals in order to convert m/z values into probable chemical structures. At the same time, data obtained by LC-MS/MS have benefited from the development of numerous chemoinformatics tools, in particular molecular networks, for their efficient annotation. For the first time, we present here the combination of MALDI-FT-ICR imaging with molecular networks from MALDI-MS/MS data directly acquired on plant tissue sections. Annotation improvements are demonstrated, paving the way for new annotation pipelines for MALDI imaging.

Structural characterization of N-acyl-homoserine lactones from bacterial quorum sensing using LC-MS/MS analyses after Paternò-Büchi derivatization in solution.

Bacterial quorum sensing is a chemical language allowing bacteria to interact through the excretion of molecules called autoinducers, like N-acyl-homoserine lactones (AHLs) produced by Gram-negative Burkholderia and Paraburkholderia bacteria known as opportunistic pathogens. The AHLs differ in their acyl-chain length and may be modified by a 3-oxo or 3-hydroxy substituent, or C = C double bonds at different positions. As the bacterial signal specificity depends on all of these chemical features, their structural characterization is essential to have a better understanding of the population regulation and virulence phenomenon. This study aimed at enabling the localization of the C = C double bond on such specialized metabolites while using significantly lower amounts of biological material. The approach is based on LC-MS/MS analyses of bacterial extracts after in-solution derivatization by a photochemical Paternò-Büchi reaction, leading to the formation of an oxetane ring and subsequently to specific fragmentations when performing MS/MS experiments. The in-solution derivatization of AHLs was optimized on several standards, and then the matrix effect of bacterial extracts on the derivatization was assessed. As a proof of concept, the optimized conditions were applied to a bacterial extract enabling the localization of C = C bonds on unsaturated AHLs.

Antiparasitic Ovalicin Derivatives from Pseudallescheria boydii, a Mutualistic Fungus of French Guiana Termites.

Social insects are in mutualism with microorganisms, contributing to their resistance against infectious diseases. The fungus Pseudallescheria boydii SNB-CN85 isolated from termites produces ovalicin derivatives resulting from the esterification of the less hindered site of the ovalicin epoxide by long-chain fatty acids. Their structures were elucidated using spectroscopic analysis and semisynthesis from ovalicin. For ovalicin, these compounds displayed antiprotozoal activities against Plasmodium falciparum and Trypanosoma brucei, with IC50 values of 19.8 and 1.1 µM, respectively, for the most active compound, i.e., ovalicin linoleate. In parallel, metabolomic profiling of a collection of P. boydii strains associated with termites made it possible to highlight this class of compounds together with tyroscherin derivatives in all strains. Finally, the complete genome of P. boydii strains was obtained by sequencing, and the cluster of potential ovalicin and ovalicin biosynthesis genes was annotated. Through these metabolomic and genomic analyses, a new ovalicin derivative named boyden C, in which the 6-membered ring of ovalicin was opened by oxidative cleavage, was isolated and structurally characterized.

Antimicrobial Activity and Synergy Investigation of Hypericum scabrum Essential Oil with Antifungal Drugs.

The chemical composition of Lebanese Hypericum scabrum essential oil (EO) was analyzed by gas chromatography (GC) and gas chromatography-mass spectrometry (GG-MS). Its antimicrobial activity was evaluated by determining its minimal inhibitory concentrations (MICs) against a Gram-negative and a Gram-positive bacterium, one yeast, and five dermatophytes. H. scabrum EO was most active on filamentous fungi (MIC values of 32-64 µg/mL). Synergy within the oil was investigated by testing each of the following major components on Trichophyton rubrum: α-pinene, limonene, myrcene, ß-pinene and nonane, as well as a reconstructed EO. The antifungal activity of the natural oil could not be reached, meaning that its activity might be due, in part, to minor constituent(s). The interactions between H. scabrum EO and commercially available antifungals were assessed by the checkerboard test. A synergistic effect was revealed in the combination of the EO with amphotericin B.

Annotation and quantification of N-acyl homoserine lactones implied in bacterial quorum sensing by supercritical-fluid chromatography coupled with high-resolution mass spectrometry.

In recent years, use of supercritical-fluid chromatography (SFC) with CO2 as the mobile phase has been expanding in the research laboratory and industry since it is considered to be a green analytical method. This technique offers numerous advantages, such as good separation and sensitive detection, short analysis times, and stability of analytes. In this study, a method for quantification of N-acyl homoserine lactones (AHLs), signaling molecules responsible for cell-to-cell communication initially discovered in bacteria, by SFC coupled with high-resolution mass spectrometry (HRMS) was developed. The SFC conditions and MS ionization settings were optimized to obtain the best separation and greatest sensitivity. The optimal analysis conditions allowed quantification of up to 30 AHLs in a single run within 16 min with excellent linearity (R2 > 0.998) and sensitivity (picogram level). This method was then applied to study AHL production by one Gram-negative endophytic bacterium, Paraburkholderia sp. BSNB-0670. Nineteen known AHLs were detected, and nine abundant HSLs were quantified. To further investigate the production of uncommon AHLs, a molecular networking approach was applied on the basis of the SFC-HRMS/MS data. This led to additional identification of four unknown AHLs annotated as N-3-hydroxydodecanoylol homoserine lactone, N-3-hydroxydodecadienoyl homoserine lactone, and N-3-oxododecenoyl homoserine lactones (two isomers).

Carneic Acids from an Endophytic Phomopsis sp. as Dengue Virus Polymerase Inhibitors.

Thirteen carneic acids were isolated from the fungal endophyte Phomopsis sp. SNB-LAP1-7-32. Their structures were identified by mass spectrometry and extensive one- and two-dimensional NMR spectroscopy and through comparison with data reported in the literature. Compounds 1-13 were investigated for their antipolymerase activities against DENV polymerase and Zika NS5. Five of them exhibited significant inhibition of dengue polymerase with IC50 values in the 10 to 20 µM range without cytotoxicity. None inhibited Zika virus NS5 protein.

Paecilosetin Derivatives as Potent Antimicrobial Agents from Isaria farinosa.

Fifty-seven entomopathogenic microorganisms were screened against human pathogens and subjected to mass spectrometry molecular networking based dereplication. Isaria farinosa BSNB-1250, shown to produce potentially novel biologically active metabolites, was grown on a large scale on potato dextrose agar, and paecilosetin (1) and five new analogues (2-6) were subsequently isolated. The structures of the new compounds were elucidated using 1D and 2D NMR. The absolute configurations of compounds 1-6 were determined using Mosher ester derivatives (1, 3, 4), comparison of experimental and calculated ECD spectra (2-4 and 6), and single-crystal X-ray diffraction analysis (5). Compounds 1 and 5 exhibited strong antibacterial activity against MSSA and MRSA with MIC values of 1-2 µg/mL.

Alsinol, an arylamino alcohol derivative active against Plasmodium, Babesia, Trypanosoma, and Leishmania: past and new outcomes.

Malaria, babesiosis, trypanosomosis, and leishmaniasis are some of the most life-threatening parasites, but the range of drugs to treat them is limited. An effective, safe, and low-cost drug with a large activity spectrum is urgently needed. For this purpose, an aryl amino alcohol derivative called Alsinol was resynthesized, screened in silico, and tested against Plasmodium, Babesia, Trypanosoma, and Leishmania. In silico Alsinol follows the Lipinski and Ghose rules. In vitro it had schizontocidal activity against Plasmodium falciparum and was able to inhibit gametocytogenesis; it was particularly active against late gametocytes. In malaria-infected mice, it showed a dose-dependent activity similar to chloroquine. It demonstrated a similar level of activity to reference compounds against Babesia divergens, and against promastigotes, and amastigotes stages of Leishmania in vitro. It inhibited the in vitro growth of two African animal strains of Trypanosoma but was ineffective in vivo in our experimental conditions. It showed moderate toxicity in J774A1 and Vero cell models. The study demonstrated that Alsinol has a large spectrum of activity and is potentially affordable to produce. Nevertheless, challenges remain in the process of scaling up synthesis, creating a suitable clinical formulation, and determining the safety margin in preclinical models.

Potent and Non-Cytotoxic Antibacterial Compounds Against Methicillin-Resistant Staphylococcus aureus Isolated from Psiloxylon mauritianum, A Medicinal Plant from Reunion Island.

With the occurrence of antibiotic-resistant Staphylococcus aureus strains, identification of new anti-staphylococcal drugs has become a necessity. It has long been demonstrated that plants are a large and diverse source of antibacterial compounds. Psiloxylon mauritianum, an endemic medicinal plant from Reunion Island, was chemically investigated for its reported biological activity against S. aureus. Aspidin VB, a phloroglucinol derivative never before described, together with Aspidin BB, were first isolated from the ethyl acetate extract of P. mauritianum leaves. Their structures were elucidated from spectroscopic data. Aspidin VB exhibited strong antibacterial activity against standard and methicillin-resistant S. aureus strains, with a minimal inhibition concentration (MIC) of 0.25 µg/mL, and no cytotoxicity was observed at 10-5 M in MRC5 cells. Due to its biological activities, Aspidin VB appears to be a good natural lead in the fight against S. aureus.

Structural Identification of Antibacterial Lipids from Amazonian Palm Tree Endophytes through the Molecular Network Approach.

A library of 197 endophytic fungi and bacteria isolated from the Amazonian palm tree Astrocaryum sciophilum was extracted and screened for antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). Four out of five antibacterial ethyl acetate extracts were also cytotoxic for the MRC-5 cells line. Liquid chromatography coupled to tandem mass spectrometry (UPHLC-HRMS/MS) analyses combined with molecular networking data processing were carried out to allow the identification of depsipeptides and cyclopeptides responsible for the cytotoxicity in the dataset. Specific ion clusters from the active Luteibacter sp. extract were also highlighted using an MRSA activity filter. A chemical study of Luteibacter sp. was conducted leading to the structural characterization of eight fatty acid exhibiting antimicrobial activity against MRSA in the tens of µg/mL range.

Characterization, Diversity, and Structure-Activity Relationship Study of Lipoamino Acids from Pantoea sp. and Synthetic Analogues.

A biological evaluation of a library of extracts from entomopathogen strains showed that Pantoea sp. extract has significant antimicrobial and insecticidal activities. Three hydroxyacyl-phenylalanine derivatives were isolated from this strain. Their structures were elucidated by a comprehensive analysis of their NMR and MS spectroscopic data. The antimicrobial and insecticidal potencies of these compounds were evaluated, and compound 3 showed 67% mortality against Aedes aegypti larvae at a concentration of 100 ppm, and a minimum inhibitory concentration (MIC) of 16 µg/mL against methicillin-resistant Staphylococcus aureus. Subsequently, hydroxyacyl-phenylalanine analogues were synthesized to better understand the structure-activity relationships within this class of compounds. Bioassays highlighted the antimicrobial potential of analogues containing saturated medium-chain fatty acids (12 or 14 carbons), whereas an unsaturated long-chain fatty acid (16 carbons) imparted larvicidal activity. Finally, using a molecular networking-based approach, several close analogues of the isolated and newly synthesized lipoamino acids were discovered in the Pantoea sp. extract.

Structurally Diverse Diterpenoids from Sandwithia guyanensis.

Bioassay-guided fractionation of an EtOAc extract of the trunk bark of Sandwithia guyanensis, using a chikungunya virus (CHIKV)-cell-based assay, afforded 17 new diterpenoids 1-17 and the known jatrointelones A and C (18 and 19). The new compounds included two tetranorditerpenoids 1 and 2, a trinorditerpenoid 3, euphoractines P-W (4-11), and euphactine G (13) possessing the rare 5/6/7/3 (4-7), 5/6/6/4 (8-11), and 5/6/8 (13) fused ring skeletons, sikkimenoid E (12), and jatrointelones J-M (14-17) possessing jatropholane and lathyrane carbon skeletons, respectively. Jatrointelones J (14) and M (17) represent the first naturally occurring examples of C-15 nonoxidized lathyrane-type diterpenoids. The structures of the new compounds were elucidated by NMR spectroscopic data analysis. The relative configuration of compound 16 and the absolute configurations of compounds 3-6 and 14 were determined by single-crystal X-ray diffraction analysis. In addition, jatrointelone K (15) was chemically transformed to euphoractine T (8) supporting the biosynthetic relationships between the two types of diterpenoids. Only compound 15 showed a moderate anti-CHIKV activity with an EC50 value of 14 µM. Finally, using a molecular networking-based dereplication strategy, several close analogues of 12- O-tetradecanoylphorbol-13-acetate (TPA), one of the most potent inhibitors of CHIKV replication, were dereplicated.

Aedes aegypti Larvicidal Sesquiterpene Alkaloids from Maytenus oblongata.

Four new sesquiterpene alkaloids (1-4) with a ß-dihydroagrofuran skeleton and a new triterpenoid (5) were isolated from an ethyl acetate extract of Maytenus oblongata stems. Their structures were elucidated using 1D and 2D NMR spectroscopy as well as MS and ECD experiments. The M. oblongata stem EtOAc extract and the pure compounds isolated were tested for larvicidal activity against Aedes aegypti under laboratory conditions, and compounds 2 and 3 were found to be active.

Betulinic Acid, The First Lupane-Type Triterpenoid Isolated from Both a Phomopsis sp. and Its Host Plant Diospyros carbonaria Benoist.

Following a biological screening using a dengue replicon virus-cell-based assay, Diospyros carbonaria AcOEt extract was investigated, affording six known lupane-type triterpenoids endowed with anti-DENV-2 NS5 polymerase activity. The study of the associated microbial community of this species permitted us to identify 38 endophytes belonging to five different orders. Nine out of these 38 strains showed significant activity on the dengue replicon assay. The chemical investigation of the most active one, Phomopsis sp. SNB-LAP1-7-32, led to the isolation of betulinic acid, an anti-viral secondary metabolite isolated previously from the host plant. This result is the first example of a lupane-type triterpenoid isolated from both an endophyte and its host plant. Its presence in the Phomopsis strain may result from gene transfer and/or specific niche selection.

Essential Oils Composition and Antimicrobial Activity of Six Conifers Harvested in Lebanon.

The chemical composition and antimicrobial activity of the essential oils (EOs) of six conifers harvested in Lebanon, Abies cilicica, Cupressus sempervirens, Juniperus excelsa, Juniperus oxycedrus, Cedrus libani and Cupressus macrocarpa gold crest, were investigated. The EOs were obtained by hydrodistillation using a Clevenger-type apparatus and characterized by GC and GC/MS analyses. A principal components analysis based on Pearson correlation between essential oils chemical analyses was also conducted. The minimum inhibitory concentrations (MICs) of these essentials oils were determined against a range of bacteria and fungi responsible for cutaneous infections in human, using the broth microdilution technique. The EOs showed the most interesting bioactivity on the dermatophytes species (MIC values 32 - 64 µg/ml). Each of the major compounds of C. macrocarpa as well as an artificial reconstructed EO were tested on Trichophyton rubrum showing a contribution of the minor components to the overall activity.

Isolation, purification and chemical characterization of a new angucyclinone compound produced by a new halotolerant Nocardiopsis sp. HR-4 strain.

A halotolerant Actinobacteria strain HR-4 was isolated from a salt lake soil sample in Algerian Sahara. Analysis of 16S rDNA gene sequence showed that strain HR-4 belonged to the genus Nocardiopsis. The similarity level ranges between 97.45 and 99.20% with Nocardiopsis species and Nocardiopsis rosea being the most closely related one. Morphological, physiological and phylogenetic characteristics comparisons showed significant differences with the nearest species. These data strongly suggest that strain HR-4 represents novel species. The antimicrobial activity of strain HR-4 showed an antibacterial activity against Gram-positive bacteria as well as an antifungal one. Two major natural products including a new one were isolated from the culture broth using various separation and purification procedures. The chemical structure established on the basis of spectroscopic studies NMR and by comparing with spectroscopic data from the literature of the two compounds affirm that they are classified in the group of Angucyclinones. This is the first report of a production of this type of molecules by the genus Nocardiopsis. The new natural compound was established as (-)-7-deoxy-8-O-methyltetrangomycin with a new configuration.

Chemical Diversity and Antimicrobial Activity of Salvia multicaulis Vahl Essential Oils.

The chemical compositions and antimicrobial activities of the essential oils (EOs) of aerial parts of Salvia multicaulis Vahl, collected during the same week from two different Lebanese regions, were investigated. The EOs were obtained by hydrodistillation using a Clevenger-type apparatus and characterized by GC and GC/MS analyses. The minimum inhibitory concentrations of these EOs were determined against one Gram-negative and two Gram-positive bacteria, one yeast, and five dermatophytes using the broth microdilution technique. One EO was notably active against Staphylococcus aureus, methicillin-resistant S. aureus, and all of the Trichophyton species tested. Nerolidol was found to be the major compound in the active oil; nerolidol was also absent from the inactive oil. This study demonstrated that nerolidol shows antimicrobial activity and therefore significantly contributes to the antimicrobial potential of the oil. The chemical diversity of worldwide S. multicaulis EOs was analyzed, revealing that the EOs of this study belong to two different chemotypes found in the literature. The nerolidol chemotype appears to be restricted to Lebanon, and it can be used as antimicrobial agent against external bacterial and fungal infections.

Assessment of A Simple Compound-Saving Method To Study Insecticidal Activity of Natural Extracts and Pure Compounds Against Mosquito Larvae.

Research on natural insecticides has intensified with the spread of resistance to chemicals among insects, particularly disease vectors. To evaluate compounds, the World Health Organization (WHO) has published standardized procedures. However, those may be excessively compound-consuming when it comes to assessing the activity of natural extracts and pure compounds isolated in limited amount. As part of our work on the discovery of new mosquito larvicides from Amazonian plants, we developed a compound-saving assay in 5-ml glass tubes instead of WHO larval 100-ml cups. Comparing activity of synthetic and natural chemicals validated the glass tube assay. Raw data, lethal doses that kill 50% (LD50) and 90% (LD90) at 24 and 48 h, were highly correlated (0.68 < R2 < 0.96, P < 0.001, Pearson test) between cups and tubes. It was also established that 10 tubes (N = 50 larvae) provided the same level of sensitivity as 20 tubes (N = 100). This method proved suitable for rapid screening of natural extracts and molecules, identifying active compounds using 10 times less material than in the WHO protocol.

Antibacterial ilicicolinic acids C and D and ilicicolinal from Neonectria discophora SNB-CN63 isolated from a termite nest.

Ilicicolinic acids A, C, and D (1-3) and ilicicolinal (4) were isolated from a fungus isolated from a Nasutitermes corniger nest in French Guiana. The structures of ilicicolinic acids C and D and ilicicolinal were elucidated using 1D and 2D NMR spectroscopic data as well as MS data. Ilicicolinic acids show antibacterial activity in vitro.

Secondary Metabolites Isolated from the Amazonian Endophytic Fungus Diaporthe sp. SNB-GSS10.

We describe a Sabicea cinerea endophytic fungus closely related to Diaporthe pseudomangiferae that produces two known metabolites, mycoepoxydiene (1) and altiloxin A (2), as well as enamidin (3) and eremofortin F (4), two compounds not previously described in the literature. The structure of these four metabolites was elucidated using spectroscopic analysis, and their cytotoxic activities were measured against the human cell lines KB, MRC-5, and MDA-MB-435.