HPLC Study of Product Formed in the Reaction of NBD-Derived Fluorescent Probe with Hydrogen Sulfide, Cysteine, N-acetylcysteine, and Glutathione

Hydrogen sulfide (H2S) and its bioderivatives analogs, such as L-cysteine (L-Cys) and glutathione (GSH), are ubiquitous biological thiols in the physiological and pathological processes of living systems. Their aberrant concentration levels are associated with many diseases. Although several NBD-based fluorescence probes have been developed to detect biological thiols, the HPLC-detection of H2S, GSH, L-Cys, and N-acetylcysteine-specific products has not been described. Herein, a novel NBD-derived pro-coumarin probe has been synthesized and used to develop a new strategy for the triple mode detection of H2S and such thiols as GSH, L-Cys, and NAC. Hydrogen sulfide and those biothiols at physiological pH release fluorescent coumarin from the probe and cause a significant fluorescence enhancement at 473 nm. The appropriate NBD-derived product for H2S, L-Cys, GSH, and NAC has a different color and retention time that allows distinguishing these biological thiols meaning the probe has a great possibility in the biological application. Fluorescent imaging combined with colorimetric and HPLC detection of H2S/biothiol-specific product(s) brings a potential tool for confirming the presence of biological thiols and determining concentrations in various aqueous biological samples

Novel Boronate Probe Based on 3-Benzothiazol-2-yl-7-hydroxy-chromen-2-one for the Detection of Peroxynitrite and Hypochlorite

Derivatives of coumarin, containing oxidant-sensitive boronate group, were recently developed for fluorescent detection of inflammatory oxidants. Here, we report the synthesis and the characterization of 3-(2-benzothiazolyl)-7-coumarin boronic acid pinacol ester (BC-BE) as a fluorescent probe for the detection of peroxynitrite (ONOO–), with high stability and a fast response time. The BC-BE probe hydrolyzes in phosphate buffer to 3-(2-benzothiazolyl)-7-coumarin boronic acid (BC-BA) which is stable in the solution even after a prolonged incubation time (24 h). BC-BA is slowly oxidized by H2O2 to form the phenolic product, 3-benzothiazol-2-yl-7-hydroxy-chromen-2-one (BC-OH). On the other hand, the BC-BA probe reacts rapidly with ONOO−. The ability of the BC-BA probe to detect ONOO– was measured using both authentic ONOO– and the system co-generating steady-state fluxes of O2•– and •NO. BC-BA is oxidized by ONOO– to BC-OH. However, in this reaction 3-benzothiazol-2-yl-chromen-2-one (BC-H) is formed in the minor pathway, as a peroxynitrite-specific product. BC-OH is also formed in the reaction of BC-BA with HOCl, and subsequent reaction of BC-OH with HOCl leads to the formation of a chlorinated phenolic product, which could be used as a specific product for HOCl. We conclude that BC-BA shows potential as an improved fluorescent probe for the detection of peroxynitrite and hypochlorite in biological settings. Complementation of the fluorescence measurements by HPLC-based identification of oxidant-specific products will help to identify the oxidants detected

Synthesis and Spectroscopic Characterization of Selected Phenothiazines and Phenazines Rationalized Based on DFT Calculation

Two unique structures were isolated from the phosphorylation reaction of 10H-phenothiazine. The 5,5-dimethyl-2-(10H-phenothiazin-10-yl)-1,3,2-dioxaphosphinane 2-oxide (2a) illustrates the product of N-phosphorylation of phenothiazine. Moreover, a potential product of 2a instability, a thiophosphoric acid 2b, was successfully isolated and structurally characterized. Molecule 2a, similarly to sulfoxide derivative 3, possesses interesting phosphorescence properties due to the presence of d-pπ bonds. The X-ray, NMR, and DFT computational studies indicate that compound 2a exhibits an anomeric effect. Additionally, the syntheses of selected symmetrical and unsymmetrical pyridine-embedded phenazines were elaborated. To compare the influence of phosphorus and sulfur atoms on the structural characteristics of 10H-phenothiazine derivatives, the high-quality crystals of (4a,12a-dihydro-12H-benzo[5,6][1,4]thiazino[2,3-b]quinoxalin-12-yl)(phenyl)methanone (1) and selected phenazines 5,12-diisopropyl-3,10-dimethyldipyrido[3,2-a:3′,2′-h]phenazine (5) and 5-isopropyl-N,N,3-trimethylpyrido[3,2-a]phenazin-10-amine (6a) were obtained. The structures of molecules 1, 2a, 2-mercapto-5,5-dimethyl-1,3,2-dioxaphosphinane 2-oxide (2b), 3,7-dinitro-10H-phenothiazine 5-oxide (3), 5 and 6a were determined by single-crystal X-ray diffraction measurements

Singlet oxygen formation from photoexcited P3HT:PCBM films applied in oxidation reactions

Poly(3-hexylthiophene) thin films containing carbon-based nanostructures, i.e. fullerenes such as buckminsterfullerene (C60) or phenyl-C61-butyric acid methyl ester (PCBM), or single-walled carbon nanotubes, were investigated as heterogeneous photosensitizers producing singlet oxygen (1O2) in aerated organic solvents. Thin films were deposited on borosilicate glass using spin coating and characterized by profilometry, UV-vis, Raman and XPS. Photogeneration of 1O2 was confirmed by photooxidation of 1,3-diphenylisobenzofuran and by reaction of 1,5-dihydroxynaphthalene to juglone. The photochemical efficiency of the blends was found to depend on the carbon-based photosensitizer and can be increased by varying its concentration in the poly(3-hexylthiophene) matrix

Mitigation of NADPH Oxidase 2 Activity as a Strategy to Inhibit Peroxynitrite Formation

International audienceUsing a high-throughput screening (HTS)-compatible assays for superoxide and hydrogen peroxide, we identified potential inhibitors of NADPH oxidase (Nox2) isoform from a small library of bioactive compounds. By using multiple probes (hydroethidine, hydropropidine, Amplex Red, and coumarin boronate) with well-defined redox chemistry that form highly diagnostic marker products upon reaction with superoxide (O 2 • –), hydrogen peroxide (H 2 O 2), and peroxynitrite (ONOO –), the number of false positives was greatly decreased. Selected hits for Nox2 were further screened for their ability to inhibit ONOO – formation in activated macrophages. New diagnostic marker product for ONOO – is reported. We conclude that the newly developed HTS/ROS assays could also be used to identify potential inhibitors of ONOO – formed from Nox2-derived O 2 • – and nitric oxide synthase (NOS)-derived nitric oxide