Benzofurazan Sulfides for Thiol Imaging and Quantification in Live Cells through Fluorescence Microscopy

Thiol groups play a significant role in various cellular functions. Cellular thiol concentrations can be affected by various physiological or pathological factors. A fluorescence imaging agent that can effectively and specifically image thiols in live cells through fluorescence microscopy is desirable for live cell thiol monitoring. Benzofurazan sulfides <b>1a</b>–<b>1e</b> were synthesized and found to be thiol specific fluorogenic agents except <b>1d</b>. They are not fluorescent but form strong fluorescent thiol adducts after reacting with thiols through a sulfide-thiol exchange reaction. On the other hand, they exhibit no reaction with other biologically relevant nucleophilic functional groups such as −NH₂, −OH, or −COOH revealing the specificity for the detection of thiols. Sulfide <b>1a</b> was selected to confirm its ability to image cellular thiols through fluorescence microscopy. The compound was demonstrated to effectively image and quantify thiol changes in live cells through fluorescence microscopy using 430 and 520 nm as the excitation and emission wavelengths, respectively. The quantification results of total thiol in live cells obtained from fluorescence microscopy were validated by an high-pressure liquid chromatography/ultraviolet (HPLC/UV) total thiol assay method. The reagents and method will be of a great value to thiol redox-related research

Thiol Specific and Mitochondria Selective Fluorogenic Benzofurazan Sulfide for Live Cell Nonprotein Thiol Imaging and Quantification in Mitochondria

Cellular thiols are divided into two major categories: nonprotein thiols (NPSH) and protein thiols (PSH). Thiols are unevenly distributed inside the cell and compartmentalized in subcellular structures. Most of our knowledge on functions/dysfunctions of cellular/subcellular thiols is based on the quantification of cellular/subcellular thiols through homogenization of cellular/subcellular structures followed by a thiol quantification method. We would like to report a thiol-specific mitochondria-selective fluorogenic benzofurazan sulfide {7,7′-thiobis­(<i>N</i>-rhodamine-benzo­[c]­[1,2,5]­oxadiazole-4-sulfonamide) (TBROS)} that can effectively image and quantify live cell NPSH in mitochondria through fluorescence intensity. Limited methods are available for imaging thiols in mitochondria in live cells especially in a quantitative manner. The thiol specificity of TBROS was demonstrated by its ability to react with thiols and inability to react with biologically relevant nucleophilic functional groups other than thiols. TBROS, with minimal fluorescence, formed strong fluorescent thiol adducts (λ_ex = 550 nm, λ_em = 580 nm) when reacting with NPSH confirming its fluorogenicity. TBROS failed to react with PSH from bovine serum albumin and cell homogenate proteins. The high mitochondrial thiol selectivity of TBROS was achieved by its mitochondria targeting structure and its higher reaction rate with NPSH at mitochondrial pH. Imaging of mitochondrial NPSH in live cells was confirmed by two colocalization methods and use of a thiol-depleting reagent. TBROS effectively imaged NPSH changes in a quantitative manner in mitochondria in live cells. The reagent will be a useful tool in exploring physiological and pathological roles of mitochondrial thiols

Design, Synthesis, and Characterization of Bis(7‑(<i>N</i>‑(2-morpholinoethyl)sulfamoyl)benzo[<i>c</i>][1,2,5]oxadiazol-5-yl)sulfane for Nonprotein Thiol Imaging in Lysosomes in Live Cells

Thiols are critical to cellular structures and functions. Disturbance of cellular thiols has been found to affect cell functions and cause various diseases. Intracellularly, thiols were found unevenly distributed in subcellular organelles. Probes capable of detecting subcellular thiol density in live cells are valuable tools in determining thiols’ roles at the subcellular level. The subcellular organelle lysosome is the place where unwanted macromolecules are removed through degradation by hydrolytic enzymes. The degradation also serves as a regulation of a variety of cellular functions such as autophagy, endocytosis, and phagocytosis to maintain cellular homeostasis. Thiols are found to be involved in the lysosomal degradation process. A probe that can detect lysosomal thiols in live cells will be a valuable tool in unveiling the roles of thiols in lysosomes. We would like to report bis­(7-(N-(2-morpholinoethyl)­sulfamoyl)­benzo­[c]­[1,2,5]-oxadiazol-5-yl)­sulfane (BISMORX) as a thiol specific fluorogenic agent for live cell nonprotein thiol (NPSH) imaging in lysosomes through fluorescence microscopy. BISMORX itself shows no fluorescence and reacts readily with a NPSH to form a fluorescent thiol adduct with excitation and emission wavelengths of 380 and 540 nm, respectively. BISMORX does not react with compounds containing nucleophilic functional groups other than thiols such as −OH, −NH2, and −COOH. No reaction was observed either when BISMORX was mixed with protein thiols. BISMORX was able to image, quantify, and detect the change of NPSH in lysosomes in live cells. A colocalization experiment with LysoTracker Red DND-99 confirmed that the thiols imaged by BISMORX were indeed lysosomal thiols

2-Acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylcarbonylamino)- phenylcarbamoylsulfanyl]propionic Acid and Its Derivatives as a Novel Class of Glutathione Reductase Inhibitors

Glutathione reductase (GR) catalyzes the reduction of oxidized glutathione to reduced glutathione. The enzyme is an attractive target for the development of antimalarial agents, agents to decrease malarial drug resistance and anticancer agents. In addition, inhibition of the enzyme has been employed as a tool in research for various purposes. In this paper, we present a rational design of 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylcarbonylamino)phenylcarbamoylsulfanyl]propionic acid and its derivatives as irreversible GR inhibitors. The Ki and kinact values of 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylcarbonylamino)phenylcarbamoylsulfanyl]propionic acid, the most potent derivative of the series, are 88 μM and 0.1 min-1, respectively. Although the Ki value of the inhibitor is in the micromolar range, it is more potent than N,N-bis(2-chloroethyl)-N-nitrosourea, which is currently the most commonly employed irreversible GR inhibitor with a reported IC50 value of 646 μM. Additional attractive features of the inhibitor include its ready availability through a one-step synthesis and good solubility in both organic and aqueous solutions