2-Amino-3'-dialkylaminobiphenyl-based fluorescent intracellular probes for nitric oxide surrogate N2O3.

Fluorescent probes for nitric oxide (NO), or more frequently for its oxidized surrogate dinitrogen trioxide (N2O3), have enabled scientists to study the contributions of this signaling molecule to many physiological processes. Seeking to improve upon limitations of other probes, we have developed a family of fluorescent probes based on a 2-amino-3'-dialkylaminobiphenyl core. This core condenses with N2O3 to form benzo[c]cinnoline structures, incorporating the analyte into the newly formed fluorophore, which results in product fluorescence with virtually no background contribution from the initial probe. We varied the substituents in the core in order to optimize both the reactivity of the probes with N2O3 and their cinnoline products' fluorescence wavelengths and brightness. The top candidates were then applied to cultured cells to verify that they could respond to NO within cellular milieus, and the top performer, NO530, was compared with a "gold standard" commercial probe, DAF-FM, in a macrophage-derived cell line, RAW 264.7, stimulated to produce NO. NO530 demonstrated similar or better sensitivity and higher selectivity for NO than DAF, making it an attractive potential alternative for NO tracking in various applications.

Understanding the Selectivity of a Multichannel Fluorescent Probe for Peroxynitrite Over Hypochlorite.

Peroxynitrite is a prominent biological reactive nitrogen species from radical combination of nitric oxide and superoxide and fundamentally involved in broad spectrum physiological and pathological processes. Though redox-inert itself, peroxynitrite anion (OONO(-)) attacks various biological electrophiles to generate an array of potent 2-e(-) or 1-e(-) oxidants, which result in cell injuries. Development of fluorescent probes for peroxynitrite, free from interference from hypochlorite, has been an active endeavor of the chemical community. We previously reported a peroxynitrite probe (PN600), which could differentiate hypochlorite from peroxynitrite through a multichannel signaling mechanism. Herein, this intriguing selectivity was accounted for through a structure-reactivity relationship study. Also, this work, together with rich literature contributions, has allowed a qualitative guideline in the use of electron-rich aromatic moieties to design probes against peroxynitrite and/or hypochlorite. The viability of this guideline was further testified by development of another list of peroxynitrite selective probes.

Practical assay for nitrite and nitrosothiol as an alternative to the Griess assay or the 2,3-diaminonaphthalene assay.

Nitrite is a heavily assayed substrate in the fields of food safety, water quality control, disease diagnosis, and forensic investigation and more recently in basic biological studies on nitric oxide physiology and pathology. The colorimetric Griess assay and the fluorimetric 2,3-diaminonaphthalene (DAN) assay are the current gold standards for nitrite quantification. They are not without limitations, yet have amazingly survived 156 and 44 years, respectively, due to the lack of a practical alternative. Both assays exhibit slow detection kinetics due to inactivation of nucleophiles under strongly acidic media, require an extensive incubation time for reaction to go completion, and hence offer a limited detection throughput. By converting an intermolecular reaction of the Griess assay intramolecularly, we designed a novel probe (NT555) for nitrite detection, which displays superior detection kinetics and sensitivity. NT555 was constructed following our "covalent-assembly" probe design principle. Upon detection, it affords a gigantic bathochromic shift of the absorption spectrum and a sensitive turn-on fluorescence signal from a zero background, both of which are typical of an "assembly" type probe. Overall, NT555 has addressed various difficulties associated with the Griess and the DAN assays and represents an attractive alternative for practical applications.

A three-channel fluorescent probe that distinguishes peroxynitrite from hypochlorite.

A novel fluorescent probe for peroxynitrite, PN(600), was rationally designed on the basis of a unique fluorophore assembly approach. PN(600) is a green-emitting coumarin derivative. Upon oxidation by peroxynitrite, PN(600) is transformed into a highly fluorescent red-emitting resorufin derivative via an orange-emitting intermediate. This three-channel signaling capability enables PN(600) to differentiate peroxynitrite from other reactive oxygen and nitrogen species, including hypochlorite and hydroxyl radical. Moreover, PN(600) is membrane-permeable and compatible with common TRITC filter sets and displays low cytotoxicity. Therefore, PN(600) is a promising candidate for in vitro peroxynitrite imaging.