Sensitive Detection of Various Forms of Hydrogen Sulfide via Highly Selective Naphthalimide-Based Fluorescent Probe.

Hydrogen sulfide (H2S) is an important gasotransmitter, but only a few methods are available for real-time detection. Fluorescent probes are attractive tools for biological applications because of their high sensitivity, convenience, rapid implementation, noninvasive monitoring capability, and simplicity in fluorescent imaging of living cells and tissues. Herein, we report on a pro-fluorescent probe, NAP-Py-N3 based on naphthalimide derivative, which was found to show high selectivity toward H2S over various other analytes, including biothiols, making it feasible to detect H2S. After reaction with H2S, this probe showed rapid and significant turn-on green fluorescent enhancement at 553 nm (about 54-fold, k2 = 9.62 M-1s-1), high sensitivity (LOD: 15.5 nM), significant Stokes shift (118 nm), and it was found that the fluorescence quantum yield of fluorescence product can reach 0.36. Moreover, the probe has also been successfully applied to detect the gaseous H2S and to confirm the presence of H2S released from modern organic donors, which in recent years have been commonly used to investigate the role of H2S in biological systems. All the results indicate that this probe is excellent and highly valuable.

A New and Fast-Response Fluorescent Probe for Monitoring Hypochlorous Acid Derived from Myeloperoxidase.

Hypochlorous acid (HOCl) has been implicated in numerous pathologies associated with an inflammatory component, but its selective and sensitive detection in biological settings remains a challenge. In this report, imaging of HOCl was realized with a thiomorpholine-based probe as derivative of nitrobenzothiadiazole (NBD-S-TM). The fluorescence is based on photoinduced electron transfer by using nitrobenzothiadiazole core as a donor and thiomorpholine substituent as an acceptor. NBD-S-TM showed high sensitivity and a fast response to HOCl k = (2.6 ± 0.2) × 107 M-1s-1 with a 1:1 stoichiometry. The detection limit for HOCl was determined to be 60 nM. Furthermore, the desirable features of NBD-S-TM for the detection of HOCl in aqueous solutions, such as its reliability at physiological pH, rapid fluorescence response, and biocompatibility, enabled its application in the detection of HOCl in myeloperoxidase enzymatic system. Moreover, NBD-S-TM exhibited excellent selectivity and sensitivity for HOCl over other biologically relevant species, such as hydrogen peroxide and peroxynitrite. The fluorescent S-oxidized product (NBD-S-TSO) is only formed in the presence of HOCl. Probing with NBD-S-TM may be helpful to further the development of high throughput screening assays to monitor the activity of myeloperoxidase.

A thiomorpholine-based fluorescent probe for the far-red hypochlorous acid monitoring.

A simple thiomorpholine-based fluorescent probe was designed and synthesized by combining thiomorpholine (TM) and nitrobenzenoselenadiazoles fluorophore (NBD-Se). The thiomorpholine group quenches the fluorescence of NBD-Se efficiently through the photoinduced electron transfer (PET) effect. Hypochlorous acid (HOCl) oxidizes the NBD-Se-TM probe to its fluorescent S-oxide (NBD-Se-TSO) with a 1:1 stoichiometry. The desirable features of NBD-Se-TM for detecting HOCl in aqueous solutions, such as its high sensitivity and selectivity, reliability at physiological pH, and rapid fluorescence response, enabled its application in the detection of HOCl produced by myeloperoxidase. The results proved that NBD-Se-TM is a promising fluorescent probe that can be used in screening assays for MPO inhibitors. Its high reaction rate constant with HOCl (2k = 2.0 × 107M-1s-1) indicates the possibility of application in more complex biological systems.

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.

Fluorescent probes for monitoring myeloperoxidase-derived hypochlorous acid: a comparative study.

MPO-derived oxidants including HOCl contribute to tissue damage and the initiation and propagation of inflammatory diseases. The search for small molecule inhibitors of myeloperoxidase, as molecular tools and potential drugs, requires the application of high throughput screening assays based on monitoring the activity of myeloperoxidase. In this study, we have compared three classes of fluorescent probes for monitoring myeloperoxidase-derived hypochlorous acid, including boronate-, aminophenyl- and thiol-based fluorogenic probes and we show that all three classes of probes are suitable for this purpose. However, probes based on the coumarin fluorophore turned out to be not reliable indicators of the inhibitors' potency. We have also determined the rate constants of the reaction between HOCl and the probes and they are equal to 1.8 × 104 M-1s-1 for coumarin boronic acid (CBA), 1.1 × 104 M-1s-1 for fluorescein based boronic acid (FLBA), 3.1 × 104 M-1s-1 for 7-(p-aminophenyl)-coumarin (APC), 1.6 × 104 M-1s-1 for 3'-(p-aminophenyl)-fluorescein (APF), and 1 × 107 M-1s-1 for 4-thiomorpholino-7-nitrobenz-2-oxa-1,3-diazole (NBD-TM). The high reaction rate constant of NBD-TM with HOCl makes this probe the most reliable tool to monitor HOCl formation in the presence of compounds showing HOCl-scavenging activity.