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

Mild Chemotriggered Generation of a Fluorophore-Tethered Diazoalkane Species via Smiles Rearrangement

In situ generation of diazoalkanes under mild conditions is desired. A mechanism based on a Smiles rearrangement has been devised that releases a fluorophore-labeled unstabilized diazoalkane in the presence of various chemical triggers. Notably, the release of this diazoalkane is accompanied by an intense fluorescence turn-on, which calibrates the release of the diazoalkane. Carboxylic acids can trap this short-lived diazoalkane intermediate and yield the corresponding esters. This transformation has potential for broad 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 (<b>PN600</b>), 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

Highly Chemo- and Regioselective Vinylation of <i>N</i>‑Heteroarenes with Vinylsulfonium Salts

An efficient chemo- and regioselective <i>N</i>-vinylation of <i>N</i>-heteroarenes has been developed using vinylsulfonium salts. The reaction proceeded under mild and transition-metal-free conditions and consistently provided moderate to high yields of vinylation products with 100% <i>E</i>-stereoselectivity. This reaction is also highly chemoselective, and compatible with a variety of functional groups, such as -NHR, -NH₂, -OH, -COOH, ester, etc

Simultaneous Quantification of Hg²⁺ and MeHg⁺ in Aqueous Media with a Single Fluorescent Probe by Multiplexing in the Time Domain

Development of a molecular probe for selective detection of MeHg⁺ in the presence of Hg²⁺ is a mission impossible to accomplish. Speciation analysis of two substrates with a single kinetic trace exploiting their differential reactivity toward a single probe, i.e., multiplexing in the time domain, is a cost-effective and powerful alternative. We have developed such a probe (<b>Hg410</b>) for simultaneously quantification of Hg²⁺ and MeHg⁺ in aqueous media. <b>Hg410</b> is designed via the “covalent-assembly” approach, displays a zero background, and bears a very concise molecular construct. It has harnessed proximity-based catalysis to achieve high reactivity toward Hg²⁺ and MeHg⁺. An unprecedentedly low detection limit of ca. 4.6 pM and 160 pM was measured for Hg²⁺ and MeHg⁺, respectively

Formation of 1,4,2-Dithiazolidines or 1,3-Thiazetidines from 1,1-Dichloro-2-nitroethene and Phenylthiourea Derivatives

A method for preparation of 1,4,2-dithiazolidine or 1,3-thiazetidine heterocycles was developed by reactions of phenylthioureas with 1,1-dichloro-2-nitroethene. The solvent has a significant influence on the type of product formation. 1,4,2-Dithiazolidines were formed in the aprotic solvent chloroform, while in the protic solvent ethanol, 1,3-thiazetidines were the main products

Super-Resolution Monitoring of Mitochondrial Dynamics upon Time-Gated Photo-Triggered Release of Nitric Oxide

Nitric oxide (NO) potentially plays a regulatory role in mitochondrial fusion and fission, which are vital to cell survival and implicated in health, disease, and aging. Molecular tools facilitating the study of the relationship between NO and mitochondrial dynamics are in need. We have recently developed a novel NO donor (<b>NOD550</b>). Upon photoactivation, <b>NOD550</b> decomposes to release two NO molecules and a fluorophore. The NO release could be spatially mapped with subdiffraction resolution and with a temporal resolution of 10 s. Due to the preferential localization of <b>NOD550</b> at mitochondria, morphology and dynamics of mitochondria could be monitored upon NO release from <b>NOD550</b>

Development of Unsymmetrical Dyads As Potent Noncarbohydrate-Based Inhibitors against Human β‑<i>N</i>‑Acetyl‑d‑hexosaminidase

Human β-<i>N</i>-acetyl-d-hexosaminidase has gained much attention due to its roles in several pathological processes and been considered as potential targets for disease therapy. A novel and efficient skeleton, which was an unsymmetrical dyad containing naphthalimide and methoxyphenyl moieties with an alkylamine spacer linkage as a noncarbohydrate-based inhibitor, was synthesized, and the activities were valuated against human β-<i>N</i>-acetyl-d-hexosaminidase. The most potent inhibitor exhibits high inhibitory activity with <i>K</i>_i values of 0.63 μM. The straightforward synthetic manners of these unsymmetrical dyads and understanding of the binding model could be advantageous for further structure optimization and development of new therapeutic agents for Hex-related diseases

Structurally Rigid 9‑Amino-benzo[<i>c</i>]cinnoliniums Make Up a Class of Compact and Large Stokes-Shift Fluorescent Dyes for Cell-Based Imaging Applications

Classic fluorescent dyes, such as coumarin, naphthalimide, fluorescein, BODIPY, rhodamine, and cyanines, are cornerstones of various spectroscopic and microscopic methods, which hold a prominent position in biological studies. We recently found that 9-amino-benzo­[<i>c</i>]­cinnoliniums make up a novel group of fluorophores that can be used in biological studies. They are featured with a succinct conjugative push–pull backbone, a broad absorption band, and a large Stokes shift. They are potentially useful as a small-molecule alternative to R-phycoerythrin to pair with fluorescein in multiplexing applications

FRET-Based Mito-Specific Fluorescent Probe for Ratiometric Detection and Imaging of Endogenous Peroxynitrite: Dyad of Cy3 and Cy5

Peroxynitrite (OONO^–) is profoundly implicated in health and disease. The physiological and pathological outcome of OONO^– is related to its local concentration, and hence, a reliable OONO^– assay is highly desired. We have developed a FRET-based small-molecule fluorescent probe (<b>PNCy3Cy5</b>), harnessing the differential reactivity of Cy3 and Cy5 toward OONO^– by fine-tuning. It exhibits high detection sensitivity and yields a ratiometric fluorescent signal. We have exemplified that it can be applied in semiquantitative determination of OONO^– in living cells. Notably, it specifically localizes in mitochondria, where endogenous OONO^– is predominantly generated. Thus, <b>PNCy3Cy5</b> is a promising molecular tool for peroxynitrite biology