Two separate commercial products of kratom [Mitragyna speciosa (Korth.) Havil. Rubiaceae] were used to generate reference standards of its indole and oxindole alkaloids. While kratom has been studied for over a century, the characterization data in the literature for many of the alkaloids are either incomplete or inconsistent with modern standards. As such, full 1H and 13C NMR spectra, along with HRESIMS and ECD data, are reported for alkaloids 1–19. Of these, four new alkaloids (7, 11, 17, and 18) were characterized using 2D NMR data, and the absolute configurations of 7, 17, and 18 were established by comparison of experimental and calculated ECD spectra. The absolute configuration for the N(4)-oxide (11) was established by comparison of NMR and ECD spectra of its reduced product with those for compound 7. In total, 19 alkaloids were characterized, including the indole alkaloid mitragynine (1) and its diastereoisomers speciociliatine (2), speciogynine (3), and mitraciliatine (4); the indole alkaloid paynantheine (5) and its diastereoisomers isopaynantheine (6) and epiallo-isopaynantheine (7); the N(4)-oxides mitragynine-N(4)-oxide (8), speciociliatine-N(4)-oxide (9), isopaynantheine-N(4)-oxide (10), and epiallo-isopaynantheine-N(4)-oxide (11); the 9-hydroxylated oxindole alkaloids speciofoline (12), isorotundifoleine (13), and isospeciofoleine (14); and the 9-unsubstituted oxindoles corynoxine A (15), corynoxine B (16), 3-epirhynchophylline (17), 3-epicorynoxine B (18), and corynoxeine (19). With the ability to analyze the spectroscopic data of all of these compounds concomitantly, a decision tree was developed to differentiate these kratom alkaloids based on a few key chemical shifts in the 1H and/or 13C NMR spectra.

Current treatment options for bacterial infections are dependent on antibiotics that inhibit microbial growth and viability. These approaches result in the evolution of drug-resistant strains of bacteria. An anti-infective strategy that is less likely to lead to the development of resistance is the disruption of quorum sensing mechanisms, which are involved in promoting virulence. The goal of this study was to identify fungal metabolites effective as quorum sensing inhibitors. Three new prenylated diresorcinols (1–3), along with two known compounds, (4R)-regiolone and decarboxycitrinone, were isolated from a freshwater fungus (Helotiales sp.) from North Carolina. Their structures were assigned on the basis of HRESIMS and NMR experiments. The structure of compound 1 was confirmed via X-ray diffraction analysis, and its absolute configuration was established by TDDFT-ECD and optical rotation calculations. Compounds 1–3 suppressed quorum sensing in a clinical isolate of methicillin-resistant Staphylococcus aureus (MRSA), with IC50 values ranging from 0.3 to 12.5 μM. These compounds represent potential leads in the development of antivirulence therapeutics.

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Four new bislactones, dihydroacremonol (1), clonostachyone (2), acremodiol B (3), and acremodiol C (4), along with one known compound, hymeglusin (5), were isolated from cultures of two fungal strains (MSX59876 and MSX59260). Both strains were identified based on phylogenetic analysis of molecular data as Clonostachys spp.; yet, they biosynthesized a suite of related, but different, secondary metabolites. Given the challenges associated with elucidating the structures and configurations of bislactones, GIAO NMR calculations were tested as a complement to traditional NMR and HRESIMS experiments. Fortuitously, the enantiomer of the new natural product (4) was known as a synthetic compound, and the predicted configuration from GIAO NMR calculations (i.e., for the relative configuration) and optical rotation calculations (i.e., for the absolute configuration) matched those of the synthesis product. These results engendered confidence in using similar procedures, particularly the mixture of GIAO NMR shift calculations coupled with an orthogonal technique, to predict the configuration of 1−3; however, there were important limitations, which are discussed for each of these. The metabolites displayed antimicrobial activities, with compounds 1 and 4 being the most potent against Staphylococcus aureus with MICs of 1 and 4 μg/mL, respectively.

For over a century, researchers have cultured microorganisms together on solid supporttypically agarin order to observe growth inhibition via antibiotic production. These simple bioassays have been critical to both academic researchers that study antibiotic production in microorganisms and to the pharmaceutical industry’s global effort to discover drugs. Despite the utility of agar assays to researchers around the globe, several limitations have prevented their widespread adoption in advanced high-throughput compound discovery and dereplication campaigns. To address a list of specific shortcomings, we developed the dual-sided agar plate assay (DAPA), which exists in a 96-well plate format, allows microorganisms to compete through opposing sides of a solid support in individual wells, is amenable to high-throughput screening and automation, is reusable, and is low-cost. Herein, we validate the use of DAPA as a tool for drug discovery and show its utility to discover new antibiotic natural products. From the screening of 217 bacterial isolates on multiple nutrient media against 3 pathogens, 55 hits were observed, 9 known antibiotics were dereplicated directly from agar plugs, and a new antibiotic, demethoxytetronasin (1), was isolated from a Streptomyces sp. These results demonstrate that DAPA is an effective, accessible, and low-cost tool to screen, dereplicate, and prioritize bacteria directly from solid support in the front end of antibiotic discovery pipelines.