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The rapid advancement of biotechnology over the recent decades has amplified the importance of DNA detection and quantification assays. Many of these assays, such as gel electrophoresis, microscopy, flow cytometry, and the detection of amplification in quantitative polymerase chain reaction (qPCR), rely on the use of DNA‐binding fluorescent dyes. This article presents a comprehensive study of six Thiazole‐Orange‐based fluorescent DNA‐binding dyes: SYBR Safe, SYBR Green, Pico Green, SYTO‐16, SYTO‐9, and the benzothiazole‐based analogue (TOPhBu) of the latter. The selected DNA markers were synthesized at a 10‐milligram scale and characterised spectroscopically to quantify their fluorescence enhancement upon binding to double‐stranded DNA. The ability of the dyes to detect DNA at low concentrations was evaluated using two new metrics, absolute fluorescence enhancement (AFE) and relative fluorescence enhancement (RFE). Quantum chemical calculations shed new light on the mechanism of their fluorogenicity through modelling the excited state behaviour and DNA binding of the dyes. Their analytical performance was further tested in qPCR experiments. The experimental results of this work highlight some important differences in the sensitivity and qPCR efficiency of the studied DNA‐binding dyes which will facilitate the DNA marker selection for analytical purposes and the future development of novel DNA sensors. [ABSTRACT FROM AUTHOR]

Background: The organism-wide effects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral infection are well studied, but little is known about the dynamics of how the infection spreads in time among or within cells due to the scarcity of suitable high-resolution experimental systems. It has been reported that SARS-CoV-2 infection pathways converge at calcium influx and subcellular calcium distribution changes. Imaging combined with a proper staining technique is an effective tool for studying subcellular calcium-related infection and replication mechanisms at such resolutions. Methods: Using two-photon (2P) fluorescence imaging with our novel Ca-selective dye, automated image analysis and clustering analysis were applied to reveal titer and variant effects on SARS-CoV-2-infected Vero E6 cells. Results: The application of a new calcium sensor molecule is shown, combined with a high-end 2P technique for imaging and identifying the patterns associated with cellular infection damage within cells. Vero E6 cells infected with SARS-CoV-2 variants, D614G or B.1.1.7, exhibit elevated cytosolic calcium levels, allowing infection monitoring by tracking the cellular changes in calcium level by the internalized calcium sensor. The imaging provides valuable information on how the level and intracellular distribution of calcium are perturbed during the infection. Moreover, two-photon calcium sensing allowed the distinction of infections by two studied viral variants via cluster analysis of the image parameters. This approach will facilitate the study of cellular correlates of infection and their quantification depending on viral variants and viral load. Conclusions: We propose a new two-photon microscopy-based method combined with a cell-internalized sensor to quantify the level of SARS-CoV-2 infection. We optimized the applied dye concentrations to not interfere with viral fusion and viral replication events. The presented method ensured the proper monitoring of viral infection, replication, and cell fate. It also enabled distinguishing intracellular details of cell damage, such as vacuole and apoptotic body formation. Using clustering analysis, 2P microscopy calcium fluorescence images were suitable to distinguish two different viral variants in cell cultures. Cellular harm levels read out by calcium imaging were quantitatively related to the initial viral multiplicity of infection numbers. Thus, 2P quantitative calcium imaging might be used as a correlate of infection or a correlate of activity in cellular antiviral studies. [ABSTRACT FROM AUTHOR]

An effective, GFP‐inspired fluorescent Zn2+ sensor is developed for two–photon microscopy and related biological application that features an 8‐methoxyquinoline moiety. Excellent photophysical characteristics including a 37‐fold fluorescence enhancement with excitation and emission maxima at 440 nm and 505 nm, respectively, as well as a high two–photon cross–section of 73 GM at 880 nm are reported. Based on the experimental data, the relationship between the structure and properties was elucidated and explained backed up by DFT calculations, particularly the observed PeT phenomenon for the turn–on process. Biological validation and detailed experimental and theoretical characterization of the free and the zinc–bound compounds are presented. [ABSTRACT FROM AUTHOR]

The few commercially available chemosensors and published probes for in vitro Zn2+ detection in two-photon microscopy are compromised by their flawed spectroscopic properties, causing issues in selectivity or challenging multistep syntheses. Herein, we present the development of an effective small molecular GFP chromophore-based fluorescent chemosensor with a 2,2′-bipyridine chelator moiety (GFZnP BIPY) for Zn2+ detection that has straightforward synthesis and uncompromised properties. Detailed experimental characterizations of the free and the zinc-bound compounds within the physiologically relevant pH range are presented. Excellent photophysical characteristics are reported, including a 53-fold fluorescence enhancement with excitation and emission maxima at 422 nm and 492 nm, respectively. A high two-photon cross section of 3.0 GM at 840 nm as well as excellent metal ion selectivity are reported. In vitro experiments on HEK 293 cell culture were carried out using two-photon microscopy to demonstrate the applicability of the novel sensor for zinc bioimaging. [ABSTRACT FROM AUTHOR]

A tremendous number of solvents, either as liquids or vapors, contaminate the environment on a daily basis worldwide. Olefin metathesis, which has been widely used as high-yielding protocols for ring-opening metathesis polymerization (ROMP), ring-closing metathesis (RCM), and isomerization reactions, is typically performed in toxic and volatile solvents such as dichloromethane. In this study, the results of our systematic experiments with the Grubbs G1, G2, and Hoveyda-Grubbs HG2 catalysts proved that benzotrifluoride (BTF) can replace dichloromethane (DCM) in these reactions, providing high yields and similar or even higher reaction rates in certain cases. The ROMP of norbornene resulted not only in high yields but also in polynorbornenes with a high molecular weight at low catalyst loadings. Ring-closing metathesis (RCM) experiments proved that, with the exception of the G1 catalyst, RCM occurs with similar high efficiencies in BTF as in DCM. It was found that isomerization of (Z)-but-2-ene-1,4-diyl diacetate with the G2 and HG2 catalysts proceeds at significantly higher initial rates in BTF than in DCM, leading to rapid isomerization with high yields in a short time. Overall, BTF is a suitable solvent for olefin metathesis, such as polymer syntheses by ROMP and the ring-closing and isomerization reactions. [ABSTRACT FROM AUTHOR]

Versatile, two-step syntheses of dihydrodibenzo[ c , e ]azepines, carbazole derivatives, and other alkaloid-type drug-like scaffolds by in situ generated azomethine ylide-induced intramolecular electrocyclization reaction from commercially available materials are presented. The reaction mechanisms of transition-metal-free carbon–carbon bond formation and the role of the kinetic control, resulting in the good regioselectivity, were confirmed by theoretical calculations. [ABSTRACT FROM AUTHOR]

Since its development, the ionophore BAPTA (1,2‐bis(2‐aminophenoxy)‐ethane‐N,N,N',N'‐tetraacetic acid) has been used unchanged in calcium sensing applications. In this work we present a comprehensive experimental and theoretical study of novel alterations in the structure of BAPTA, with a focus on the systematic modification of the chain connecting the two aromatic rings of the molecule (denoted as "linker"). A bis‐(diethylamino)xantene fluorophore was also attached to the structures in a fixed position and the structure‐fluorescence response relationship of these molecules was investigated in addition. The effect of the linker's length, the number of oxygen atoms in this chain and even the removal of one of the rings was tested; these all proved to significantly alter the characteristics of the compounds. For example, it was found that the second aromatic ring of BAPTA is not essential for the turn‐on of the fluorescence. We also demonstrated that successful sensing can be realized even by replacing the chain with a single oxygen atom, which suggests the availability of a new calcium binding mode of the chelator. The reliable turn‐on characteristic, the steep Ca2+ fluorescence titration curve and the intense fluorescence emission combine to make this compound a prospective candidate as a calcium sensing molecular probe in diagnostic neurobiological applications. [ABSTRACT FROM AUTHOR]

The first enzyme-catalyzed kinetic resolution of tert-butyl-3-hydroxy-4-phenylpyrrolidine-1-carboxylate is presented. Enzyme, solvent and temperature optimization resulted in a new resolution method with E = 40 enantioselectivity. The acetate derivative of the (+)-(3 S,4 R) enantiomer formed while the (−)-(3R,4S) isomer remained intact. Very good enantioselectivities (E > 200) were achieved in the enzyme-catalyzed alcoholysis of the racemic acetate in i-propanol and t-butanol where the (+)-(3 S,4 R) enantiomer was prepared in pure form (ee > 99.7%). Absolute configuration of the (−)-(3 R,4 S)-enantiomer was determined by single crystal X-ray diffraction method. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

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