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Natural product congeners serve a useful role in the understanding of natural product biosynthesis and structure-activity relationships. A minor congener with superior activity, selectivity, and modifiable functional groups could serve as a more effective lead structure and replace even the original lead molecule that was used for medicinal chemistry modifications. Currently, no effective method exists to discover targeted congeners rapidly, specifically, and selectively from producing sources. Herein, a new method based on liquid-chromatography tandem-mass spectrometry combination is evaluated for targeted discovery of congeners of platensimycin and platencin from the extracts of Streptomyces platensis . By utilizing a precursor-ion searching protocol, tandem mass spectrometry not only confirmed the presence of known congeners but also provided unambiguous detection of many previously unknown congeners of platensimycin and platencin. This high-throughput and quantitative method can be rapidly and broadly applied for dereplication and congener discovery from a variety of producing sources, even when the targeted compounds are obscured by the presence of unrelated natural products.

Strain prioritization for drug discovery aims at excluding redundant strains of a collection in order to limit the repetitive identification of the same molecules. In this work, we wanted to estimate what can be unexploited in terms of the amount, diversity, and novelty of compounds if the search is focused on only one single representative strain of a species, taking Streptomyces lunaelactis as a model. For this purpose, we selected 18 S. lunaelactis strains taxonomically clustered with the archetype strain S. lunaelactis MM109 T . Genome mining of all S. lunaelactis isolated from the same cave revealed that 54% of the 42 biosynthetic gene clusters (BGCs) are strain specific, and five BGCs are not present in the reference strain MM109 T . In addition, even when a BGC is conserved in all strains such as the bag / fev cluster involved in bagremycin and ferroverdin production, the compounds produced highly differ between the strains and previously unreported compounds are not produced by the archetype MM109 T . Moreover, metabolomic pattern analysis uncovered important profile heterogeneity, confirming that identical BGC predisposition between two strains does not automatically imply chemical uniformity. In conclusion, trying to avoid strain redundancy based on phylogeny and genome mining information alone can compromise the discovery of new natural products and might prevent the exploitation of the best naturally engineered producers of specific molecules.

As part of our research to discover novel bioactive natural products from marine microorganisms, five bagremycin analogues, including the previously unreported bagremycins F ( 1 ) and G ( 2 ), were isolated from a marine actinomycete Streptomyces sp. ZZ745. The structures of these compounds were determined by means of NMR spectroscopic analysis, HRESIMS data, and optical rotation. Both bagremycins F ( 1 ) and G ( 2 ) showed antibacterial activity against Escherichia coli , with MIC values of 41.8 and 61.7 μM, respectively.

A practical solid-phase extraction (SPE) method coupled with liquid chromatography/tandem mass spectrometry (LC-MS/MS) has been developed for the determination of the fish anesthetic MS-222 in water. Water samples were concentrated and purified using three SPE cartridges of different specifications. Elution curves of MS-222 were constructed using various methanol-water solutions on the different cartridges, and SPE conditions were optimized in accordance with the elution curves. The mobile phase containing methanol and 0.1% formic acid solution with a linear gradient elution was utilized to separate MS-222 on a C18 column. Detection was carried out by a triple-quadrupole mass spectrometry with an electrospray ion source in positive mode. Recoveries of three MS-222 fortified levels of 0.05, 0.5 and 5 μg/L ranged of 82.6-101% with relative standard deviations (RSDs) below 9.36%. The limit of detection (LOD) and limit of quantification (LOQ) of MS-222 were 0.01 μg/L and 0.03 μg/L, respectively. This method was satisfactorily applied to the determination of MS-222 in actual water samples collected from aquatic product transportation vehicles or from the natural water catchments., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Streptomyces platensis MA7327 is a bacterium producing interesting antibiotics, which act by the novel mechanism of inhibiting fatty acid biosynthesis. The antibiotics produced by this actinomycete are platensimycin and platencin plus some minor related antibiotics. Platensimycin and platencin have activity against antibiotic-resistant bacteria such as methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus; they also lack toxicity in animal models. Platensimycin also has activity against diabetes in a mouse model. We have been interested in studying the effects of primary metabolites on production of these antibiotics in our chemically defined production medium. In the present work, we tested 32 primary metabolites for their effect. They included 20 amino acids, 7 vitamins and 5 nucleic acid derivatives. Of these, only l-aspartic acid showed stimulation of antibiotic production. We conclude that the stimulatory effect of aspartic acid is due to its role as a precursor involved in the biosynthesis of aspartate-4-semialdehyde, which is the starting point for the biosynthesis of the 3-amino-2,4-dihydroxy benzoic acid portion of the platensimycin molecule.

Analytical methods based on multiplug filtration cleanup coupled with pulse glow discharge-ion mobility spectrometry and liquid chromatography tandem mass spectrometry were developed for the analysis of tricaine mesylate residue in fish and fish-raising water samples. A silica fiber holder and an appropriate new interface were designed to make the direct introduction of the fiber into the pulse glow discharge-ion mobility spectrometry introduction mechanism. The multiplug filtration cleanup method with adsorption mixtures was optimized for the determination of tricaine mesylate in fish samples. Good linear relationships were obtained by the two methods. For fish samples, limits of detection were 6 and 0.6 μg/kg by ion mobility spectrometry and liquid chromatography with tandem mass spectrometry, respectively. The matrix effect of the established liquid chromatography tandem mass spectrometry method was negligible for fish samples but that of the ion mobility spectrometry method was not. The two methods were compared. The ion mobility spectrometry system could be used a rapid screening tool on site with the advantage of rapidity, simplicity, and portability, and the liquid chromatography tandem mass spectrometry system could be used for validation in laboratory conditions with the advantage of lower limit of detection, stability, and precision., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The retention behavior of fifteen isomeric hydroxy- and aminobenzoic acids in zwitterionic hydrophilic interaction chromatography was studied using a sulfobetaine phase (ZIC-HILIC). By an inspection of their molecular structures, the retention was related to the number, the position and hydrogen bond properties of the functional groups. The effect of the chromatographic conditions was analyzed in order to investigate the retention mechanism of the stationary phase. The increased retention observed for negative charged compounds when the mobile phase pH decreased was ascribed to a diminishing of the electrostatic repulsion with the underivatized silanol groups. Also the salt buffer concentration in the mobile was proved to have a great influence in the modulation of the electrostatic interactions. However, the retention behavior of the benzoic acids was not described by conventional ion-exchange models. Subsequently, a systematical analysis of partition, adsorption, and hydrophilic chromatographic models was presented. The results from the fittings indicated that partition processes govern mainly the ZIC-HILIC separation, but also adsorption processes via hydrogen bonds occurred for hydrogen donor analytes. Finally, the influence of the chromatographic conditions on the water enriched layer in which partition takes place has been evaluated by the elution behavior of toluene., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Platensimycin (1a) and platencin (2) are inhibitors of FabF and FabF/H bacterial fatty acid synthase. The discovery of natural congeners is an approach that can render a better understanding of the structure-function relationships of complex natural products. The isolation and structure elucidation of nine new congeners (11-20) of platensimycin and platencin are described from a fermentation broth of Streptomyces platensis. These hydroxylated congeners are likely derived by cytochrome P450 oxidation of the terpenoid units post-cyclization. Polar groups in the terpenoid portion of the molecule produce negative interactions with the hydrophobic pocket of FabF, resulting in poor activities. However, the discovery of these compounds serves an important purpose, not only to understand structure-function relationships, which cannot be easily accessed by chemical modification, but also to provide access to compounds that could be used for structural identification/confirmation of the oxidative trace metabolites produced in vivo during animal experiments.

For decades, microbial natural products have been one of the major sources of novel drugs for pharmaceutical companies, and today all evidence suggests that novel molecules with potential therapeutic applications are still waiting to be discovered from these natural sources, especially from actinomycetes. Any appropriate exploitation of the chemical diversity of these microbial sources relies on proper understanding of their biological diversity and other related key factors that maximize the possibility of successful identification of novel molecules. Without doubt, the discovery of platensimycin has shown that microbial natural products can continue to deliver novel scaffolds if appropriate tools are put in place to reveal them in a cost-effective manner. Whereas today innovative technologies involving exploitation of uncultivated environmental diversity, together with chemical biology and in silico approaches, are seeing rapid development in natural products research, maximization of the chances of exploiting chemical diversity from microbial collections is still essential for novel drug discovery. This work provides an overview of the integrated approaches developed at the former Basic Research Center of Merck Sharp and Dohme in Spain to exploit the diversity and biosynthetic potential of actinomycetes, and includes some examples of those that were successfully applied to the discovery of novel antibiotics.

Platensimycin and platencin are inhibitors of FabF and FabF/H bacterial fatty acid synthesis enzymes, respectively. Discovery of natural congeners provides one of the ways to understand the relationship of chemical structure and biological function. Efforts to discover the natural analogs of platensimycin by chemical screening led to the isolation of platensimycin B(4), a glucoside congener of platensimycin. This analog showed significantly attenuated activity and critically defined the limited binding space around the aromatic ring and established the importance of the free phenolic and carboxyl group for the activity.

Natural products continue to serve as one of the best sources for discovery of antibacterial agents as exemplified by the recent discoveries of platensimycin and platencin. Chemical modifications as well as discovery of congeners are the main sources for gaining knowledge of structure-activity relationship of natural products. Screening for congeners in the extracts of the fermentation broths of Streptomyces platensis led to the isolation of platencin A(1), a hydroxy congener of platencin. The hydroxylation of the tricyclic enone moiety negatively affected the antibacterial activity and appears to be consistent with the hydrophobic binding pocket of the FabF. Isolation, structure, enzyme-bound structure and activity of platencin A(1) and two other congeners have been described.

Platensimycins B(1)-B(3) are natural congeners of platensimycin with modest to significant changes in the benzoic acid portion of the molecule, leading to attenuation in the biological activities and thus confirming the significance of the free carboxylate for the potent activity.

The Biflow, a new isoelectric trapping instrument was designed to obtain a narrow DeltapI fraction from a complex feed in one step. The Biflow contains two identical separation units, each unit houses: an anode and cathode compartment, an anodic and cathodic membrane, an anodic and cathodic separation compartment, and a separation membrane. The separation units are connected to independent power supplies. The anodic membranes in Units 1 and 2 typically buffer at the same pH value and so do the cathodic membranes. The separation membranes in Units 1 and 2 buffer at different pH values, these determine the pI range (DeltapI) of the product. The cathodic separation compartments in Units 1 and 2 contain the feed and harvest streams. The two anodic separation compartments, connected through an electrically insulating air gap, form the transfer loop through which the transfer stream is recirculated between Units 1 and 2. Ampholytic components in the feed, with pI values lower than the pH of the buffering membrane in Unit 1, pass into the transfer stream and are shuttled into Unit 2. In Unit 2, components in the transfer stream which have pI values higher than the pH of the buffering membrane in Unit 2, pass into the harvest stream. This double transfer of the target component, oppositely directed, guarantees the complete exclusion of products outside the desired DeltapI range from the harvest stream. The utility of the Biflow unit was demonstrated by isolating carnosine from a mixture of UV-absorbing ampholytes and ovalbumin isoforms as well as 4.4

Fatty acids are essential for survival of bacteria and are synthesized by a series of enzymes including the elongation enzymes, beta-ketoacyl acyl carrier protein synthase I/II (FabF/B). Inhibition of fatty acid synthesis is one of the new targets for the discovery and development of antibacterial agents. Platensimycin (1a) is a novel broad spectrum Gram-positive antibiotic produced by Streptomyces platensis. It was discovered by target-based whole-cell screening strategy using antisense differential sensitivity assay. It inhibits bacterial growth by selectively inhibiting condensing enzyme FabF of the fatty acid synthesis pathway and was isolated by a two-step process, a capture step followed by reversed-phase HPLC. The structure was elucidated by 2D NMR methods and confirmed by X-ray crystallographic analysis of a bromo derivative. It was determined that potential reactivity of the enone moiety does not play a key role in the biological activity of platensimycin. However, cyclohexenone ring conformation renders for the stronger binding interaction with the enzyme. The isolation, structure elucidation, derivatization, and biological activity of 6,7-dihydroplatensimycin are described.

Two novel secondary metabolites, bagremycin A (2) and B (3), were detected in the culture filtrate of Streptomyces sp. Tü 4128 by HPLC-diode-array screening. They are phenol esters of 3-amino-4-hydroxybenzoic acid with a derivative of p-coumaric acid and show a moderate activity against gram-positive bacteria and some fungi.

Two new biotransformation products, N-acetyl-m-aminobenzoic acid and N-formyl-m-aminobenzoic acid were isolated from cell suspension cultures of Solanum laciniatum following administration of m-aminobenzoic acid, and their structures were elucidated using one- and two-dimensional 1H- and 13C-NMR data.