Stable Isotope Labeling of Prodiginines and Serratamolides Produced by Serratia marcescens Directly on Agar and Simultaneous Visualization by Matrix-Assisted Laser Desorption/Ionization Imaging High-Resolution Mass Spectrometry.

Matrix-assisted laser desorption/ionization imaging high-resolution mass spectrometry (MALDI-imaging-HRMS) is an important technique for visualizing the spatial distribution of compounds directly on the surface of organisms such as microorganisms, insects, plants, animals, and human tissues. However, MALDI-imaging-HRMS and the stable isotope labeling approach have never been combined for the detection and simultaneous visualization of labeled and unlabeled compounds, their analogues and derivatives, as well as their precursors. Herein, we present a methodology that labels microbial secondary metabolites directly on agar with stable isotopes and allows concurrent spatial distribution analyses by MALDI-imaging-HRMS. Using a thin film of labeled agar supplemented with [1-13C]-l-proline, [methyl-D3]-l-methionine, 15NH4Cl, or [15N]-l-serine overlaid on unlabeled agar, we demonstrate the incorporation of labeled precursors into prodiginines and serratamolides produced by an endophytic bacterium, Serratia marcescens, by MALDI-imaging-HRMS and HPLC-HRMS. Further, we show the incorporation of CD3 into prodigiosin as well as its characteristic fragments directly by MALDI-imaging-HRMS2. Our methodology has several advantages over currently existing techniques. First, both labeled and unlabeled compounds can be visualized simultaneously in high spatial resolution along with their labeled and unlabeled precursors. Second, by using a thin film of labeled agar, we utilize minimum amounts of expensive labeled compounds (1-3 mg) ensuring a cost-effective method for investigating biosynthetic pathways. Finally, our method allows in situ visualization and identification of target and nontarget compounds without the need of isolating the compounds. This is important for compounds that are produced by microorganisms in low, physiologically, or ecologically relevant concentrations.

Cytotoxic and genotoxic properties of artathomsonine, a new oxoberberine alkaloid from Artabotrys thomsonii (annonaceae).

A phytochemical investigation of the liana of Artabotrys thomsonii led to the isolation of a new oxoberberine alkaloid, 2,10-dihydroxy-3,9-dimethoxy-8-oxo-protoberberine (7), along with six known compounds. Their chemical structures were elucidated by 1 D and 2 D NMR spectroscopic methods and HRESI-MSn data analysis. Compounds 4 and 7 were selected for further in vitro investigations. In accordance with expectations from their chemical structures, compounds 7 and 4 showed a clear antioxidant activity in a cell-free assay, with compound 7 being 7-fold more active than 4. Cytotoxicity, cytostatic and genotoxic effects only occurred at high micromolar concentrations of 50 µM or more. Compound 7 was slightly less effective than compound 4. A low micromolar concentration of 10 µM did not cause any damaging cellular effects but showed potential for a protection against the micronucleus-inducing effect of reactive oxygen species hydrogen peroxide, although not to a significant extent.

Isomeric stability of indaziflam and major degradation products in the environment.

Understanding the isomeric behavior of active ingredients in the soil and water environment is the first and a major part of deriving an exposure assessment. Whilst a variety of approaches have been taken previously, with the new regulatory framework for the risk assessment of isomeric plant protection compounds recently published by EFSA, (European Food Safety Authority) there will in future be a more consistent approach which has been taken here. For indaziflam (IAF), the alkylazine, cross spectrum residual herbicide which has a cellulose biosynthesis inhibition mode of action, there was no published data on the isomeric degradation behavior in soil and water. The results of measuring the isomeric stability of [14C]-radiolabeled 437-IAF, the major stereoisomer of indaziflam (AE 1170437, [1R,2S,6R] configuration) during its degradation in an aerobic soil metabolism study with four EU soils, an aerobic aquatic metabolism study with two natural water/sediment test systems, as well as an aqueous photolysis study are reported. To sum up, it was shown that in the different environmental conditions under abiotic as well as biotic degradation processes, indaziflam was not subject to isomeric interconversion to diastereoisomers 435-IAF (RRR), 438-IAF (RSS), or 439-IAF (SSR). Thus, all three chiral centers of indaziflam can be considered isomerically stable. In addition, no isomeric interconversion was observed at the 1-fluoroethyl position for the major degradation products IAF-indanone and IAF-carboxylic acid to the RSS-configuration as well as IAF-diaminotriazine from the R- to the S-configuration.

Spatial-temporal profiling of prodiginines and serratamolides produced by endophytic Serratia marcescens harbored in Maytenus serrata.

An endophytic bacterium, Serratia marcescens MSRBB2, isolated from inner bark of a Cameroonian Maytenus serrata plant, was subjected to the OSMAC (One Strain Many Compounds) approach and metabolic profiling using HPLC-HRMSn. We identified 7 prodiginines along with 26 serratamolides. Their biosynthetic pathways were elucidated by feeding with labeled precursors in combination with HRMSn. Dual-culture confrontation/restriction assays of the bacterial endophyte were devised with coexisting fungal endophytes (Pestalotiopsis virgatula, Aspergillus caesiellus and Pichia spp.) as well as with unrelated, non-endophytic fungi belonging to the same genera. The assays were combined with scanning electron microscopy (SEM) as well as matrix-assisted laser desorption ionization imaging high-resolution mass spectrometry (MALDI-imaging-HRMS) for visualizing, both in high spatial and temporal resolution, the distribution and interplay of the compounds during microbial interactions. We demonstrated the effect of prodigiosin produced by endophytic S. marcescens MSRBB2 as an allelochemical that specifically inhibits coexisting endophytic fungi. Our results provide new insights into the physiological and ecological relevance of prodiginines and serratamolides within the context of allelopathy and chemical defense interaction occurring between coexisting endophytes harbored in M. serrata.

Spatial profiling of maytansine during the germination process of Maytenus senegalensis seeds.

The ecological role of maytansine, an important antineoplastic and antimicrobial compound with high cytotoxicity, particularly as a chemical defense compound has remained elusive since its discovery in the 1970s in Maytenus and Putterlickia plants. In the present study, we have used MALDI-imaging-HRMS to visualize the occurrence as well as spatial and temporal distribution of maytansine in a Maytenus senegalensis plant, seeds obtained from the mother plant during seeding stage, through the germination of the seeds, and finally up to the establishment of seedlings (or daughter plants). Although the mother plant was devoid of maytansine, the bioactive compound was found to be distributed in the cotyledons and the endosperm of the seeds with an augmented accretion towards the seed coat. Furthermore, maytansine was always detected in the emerging seedlings, particularly the cortex encompassing the radicle, hypocotyl, and epicotyl. The typical pattern of accumulation of maytansine not only in the seeds but also during germination provides a proof-of-concept that M. senegalensis is ecologically primed to trigger the production of maytansine in vulnerable tissues such as seeds during plant reproduction. By utilizing maytansine as chemical defense compound against predators and/or pathogens, the plant can ensure viability of the seeds and successful germination, thus leading to the next generation of daughter plants.

Occurrence and spatial distribution of maytansinoids in Putterlickia pyracantha, an unexplored resource of anticancer compounds.

Maytansinoids possess remarkable antibiotic activities along with high cytotoxicity, many of which are currently used (or in clinical trials) in the treatment of breast cancer. Celastraceous plants and their associated microorganisms serve as an important resource of maytansinoids. Here, we report the occurrence and structural elucidation of several maytansinoids in Putterlickia pyracantha plants bioprospected in South Africa. In addition to maytansine, which is already known to be present in this species, we show the presence of maytanprine, maytanbutine, maytanvaline, normaytancyprine and an abundant maytansine precursor in different tissues using high-resolution mass spectrometry. Furthermore, we identified two new hydroxylated maytansinoids by HRMS(2) analyses. We also employed MALDI-imaging-HRMS to study the spatial distribution and localization of the maytansinoids within the different plant tissues. On the one hand, the fragmentation pathways of the maytansinoids we report herein using HRMS(n) will allow quick identification of these compounds in the future without isolating from the natural resources. On the other hand, MALDI-imaging-HRMS revealed insights into the plausible ecological roles and biosynthetic pathways of these compounds in P. pyracantha plants.

Erylivingstone A-C with antioxidant and antibacterial activities from Erythrina livingstoniana.

The chemical study of Erythrina livingstoniana has led to the isolation of three new flavanones namely 5,7,3'-trihydroxy-4'-methoxy-5'-formylflavanone (erylivingstone A) (1), 5,7,3'-trihydroxy-5'-(2-hydroxy-3-methylbut-3-enyl)-4'-methoxyflavanone (erylivingstone B) (2) and 5,7,3'-trihydroxy-5'-(3-hydroxy-3-methyl-trans-but-1-enyl)-4'-methoxyflavanone (erylivingstone C) (3) together with three known compounds (4-6). Their structures were elucidated on the basis of NMR data, HRMS(n) fragmentation pathway and by comparison with literature data. We evaluated the antibacterial efficacies and free-radical scavenging potential of the isolated compounds (1-6). The typical environmental strains of Gram-positive Bacillus subtilis, Gram-negative Escherichia coli, as well as against the clinically important Staphylococcus aureus, Streptococcus pyogenes and E. coli (risk-group 2) were used for the antibacterial assay. Compounds 5 and 6 exhibited the most pronounced efficacy against tested environmental bacteria as well as against the pathogenic strain of E. coli. Compound 3 was also quite active against these three bacterial strains. The isolated compounds showed weak radical scavenging properties with compound 6 being the most active, followed by compounds 2, 3 and 5.

Anti-inflammatory tirucallane triterpenoids from Anopyxis klaineana Pierre (Engl.), (Rhizophoraceae).

Phytochemical investigation of the stem bark extract of Anopyxis klaineana was carried out by various chromatographic techniques resulting in the isolation and characterization of three new tirucallane triterpenoids, namely 3,23-dioxotirucalla-7,24-dien-21-oic acid (1), 3,4-secotirucalla-23-oxo-4(28)7,24-trien-3,21-dioic acid (2) and 3,4-secotirucalla-4-hydroxy-23-oxo-7,24-diene-3,21-dioic acid-21-methyl ester (3), along with nine known compounds (4-12). The structural elucidation of the compounds was performed by means of high-resolution mass spectrometry (HRMS(n)), nuclear magnetic resonance (NMR), and by comparison to literature data. Although none of the isolated compounds showed antibacterial efficacy against selected environmental and clinically important pathogenic Gram-positive and -negative bacteria, they demonstrated moderate DPPH free radical scavenging properties. Furthermore, compounds 1 and 7 exhibited remarkable anti-inflammatory potential in a prostaglandin E2 (PGE2) competitive enzyme immunoassay with IC50 values of 3.63 µM and 10.23 µM, respectively.