Proposal for structure revision of pinofuranoxin A through total syntheses of stereoisomers.

The relative configuration of the epoxide functionality in pinofuranoxin A (1), α-alkylidene-ß-hydroxy-γ-methyl-γ-butyrolactone with trans-epoxy side chain isolated by Evidente et al. in 2021, was revised by DFT-based spectral reinvestigations and stereo-controlled synthesis. The present investigation demonstrates the difficulty of the configurational elucidation of the stereogenic centers on the conformationally flexible acyclic side-chains. Sharpless's enantioselective epoxidations and dihydroxylations were quite effective in the reinvestigations of the configurations. As our syntheses made all diastereomers available, these would be quite effective in the next structure-biological activity relationship studies.

Development of a Paper-Based Luminescence Bioassay for Therapeutic Monitoring of Aminoglycosides: a Proof-of-Concept Study.

Aminoglycosides are widely used antibiotics that bind to the bacterial 30S ribosomal subunit to inhibit translation. Owing to their adverse side effects and narrow therapeutic index, monitoring blood levels of aminoglycosides is important to maximize their effectiveness and minimize their toxicity. Current monitoring techniques require a well-equipped diagnostic laboratory. The present study aimed to present a proof-of-concept for a simple, low-cost biochemical assay utilizing a paper platform for the detection of serum/whole blood aminoglycosides. A paper-based bioassay chip for the assay was developed by spotting and freeze-drying cell-free transcription/translation reaction machinery for a luminescent reporter protein (NanoLuc) within an array of wax circles printed on filter paper. The paper-based chip could be used to quantify serum/whole blood aminoglycosides within clinically relevant concentrations in 30-60 min by spotting minimal volumes of samples, followed by the NanoLuc substrate, in the wax circles and detecting the associated changes in luminescence signals, using a simple digital camera. Furthermore, a one-pot assay in which cell-free transcription/translation reaction machinery and NanoLuc substrate are mixed in advance and embedded in paper could be used to detect an aminoglycoside in serum. Overall, our paper-based bioassay can potentially provide a basic platform for the simple and low-cost therapeutic monitoring of aminoglycosides, especially in resource-limited regions.

Paper-based luminescence bioassay method embedding a sequence of enzymatic reactions to detect sulfonamide groups.

Sulfonamide residue in foodstuffs and the environment is a serious global concern for their contribution to the occurrence of antibiotic-resistant bacteria, especially in developing countries. Here, we describe a novel, simple, and low-cost bioassay for sulfonamides, which has high potential versatility for use in low-resource settings. The bioassay method is based on a purpose-built luminescent assay reaction that detects sulfonamide groups. The luminescent assay reaction comprises dihydropteroate synthase, a target enzyme of sulfonamides, and luminescent pyrophosphate detection reagent, which triggers a sequence of biomolecular reactions that convert sulfonamides to emit luminescence. The novel assay detected at least six different sulfonamides with an estimated limit of detection of <25 ng ml-1 in a solution-phase assay using a microplate reader. More importantly, the luminescent assay reaction functioned even after spotting and freeze-drying on a wax pattern-printed paper platform. The paper-embedded luminescent assay reaction showed response signals to sulfadiazine within 30 min at a limit of detection similar to that of the solution-phase assay using a microplate reader. The signal could be recorded using a digital camera in the dark and required no other laboratory infrastructure, freeing the assay from the constraints of a well-fitted laboratory.

Paper-based colorimetric biosensor for antibiotics inhibiting bacterial protein synthesis.

Due to the presence of antibiotics in environmental water and their potential influence on the occurrence of antibiotic-resistant bacteria, development of a detection method suitable for the screening of environmental water for antibiotics is required. In this study, we developed a simple colorimetric paper-based biosensor based on a novel principle for the detection of antibiotics inhibiting bacterial protein synthesis, including aminoglycosides, tetracycline, chloramphenicol, and macrolides. This biosensor is based on the detection of a color change induced by ß-galactosidase, which is synthesized on freeze-dried paper discs containing an in vitro transcription/translation system. When a water sample without antibiotics is applied to the paper discs, ß-galactosidase can be synthesized, and it hydrolyzes a colorimetric substrate, resulting in a color change from yellow to purple. By contrast, in the presence of antibiotics, the color change can be hampered due to an inhibition of ß-galactosidase synthesis. We investigated the effect of the incubation temperature and pH of water samples and confirmed that the paper discs showed the color change to purple in the ranges of 15-37°C and pH 6-10. We observed concentration-dependent color variations of the paper discs by the naked eye and further estimated detection limits to be 0.5, 2.1, 0.8, and 6.1 µg/mL for paromomycin, tetracycline, chloramphenicol, and erythromycin, respectively, using digitized pictures. The paper-based biosensor proved to detect 0.5 µg/mL paromomycin, spiked in real environmental water samples, by the naked eye.