A Ratiometric Fluorescent Probe for Hypochlorite and Lipid Droplets to Monitor Oxidative Stress.

Mitochondria are valuable subcellular organelles and play crucial roles in redox signaling in living cells. Substantial evidence proved that mitochondria are one of the critical sources of reactive oxygen species (ROS), and overproduction of ROS accompanies redox imbalance and cell immunity. Among ROS, hydrogen peroxide (H2O2) is the foremost redox regulator, which reacts with chloride ions in the presence of myeloperoxidase (MPO) to generate another biogenic redox molecule, hypochlorous acid (HOCl). These highly reactive ROS are the primary cause of damage to DNA (deoxyribonucleic acid), RNA (ribonucleic acid), and proteins, leading to various neuronal diseases and cell death. Cellular damage, related cell death, and oxidative stress are also associated with lysosomes which act as recycling units in the cytoplasm. Hence, simultaneous monitoring of multiple organelles using simple molecular probes is an exciting area of research that is yet to be explored. Significant evidence also suggests that oxidative stress induces the accumulation of lipid droplets in cells. Hence, monitoring redox biomolecules in mitochondria and lipid droplets in cells may give a new insight into cell damage, leading to cell death and related disease progressions. Herein, we developed simple hemicyanine-based small molecular probes with a boronic acid trigger. A fluorescent probe AB that could efficiently detect mitochondrial ROS, especially HOCl, and viscosity simultaneously. When the AB probe released phenylboronic acid after reacting with ROS, the product AB-OH exhibited ratiometric emissions depending on excitation. This AB-OH nicely translocates to lysosomes and efficiently monitors the lysosomal lipid droplets. Photoluminescence and confocal fluorescence imaging analysis suggest that AB and corresponding AB-OH molecules are potential chemical probes for studying oxidative stress.

Activity-Based Fluorescent Probes for Sensing and Imaging of Reactive Carbonyl Species (RCSs).

This review explains various strategies for developing fluorescent probes to detect reactive carbonyl species (RCS). There are several mono and diacarbonyls among 30 varieties of reactive carbonyl species (RCSs) so far discovered, which play pivotal roles in pathological processes such as cancer, neurodegenerative diseases, cardiovascular disease, renal failure, and diabetes mellitus. These RCSs play essential roles in maintaining ion channel regulation, cellular signaling pathways, and metabolisms. Among RCSs, carbon monoxide (CO) is also utilized for its cardioprotective, anti-inflammatory, and anti-apoptotic effects. Fluorescence-based non-invasive optical tools have come out as one of the promising methods for analyzing the concentrations and co-localizations of these small metabolites. There has been a tremendous eruption in developing fluorescent probes for selective detection of specific RCSs within cellular and aqueous environments due to their high sensitivity, high spatial and temporal resolution of fluorescence imaging. Fluorescence-based sensing mechanisms such as intramolecular charge transfer (ICT), photoinduced electron transfer (PeT), excited-state intramolecular proton transfer (ESIPT), and fluorescence resonance energy transfer (FRET) are described. In particular, probes for dicarbonyls such as methylglyoxal (MGO), malondialdehyde (MDA), along with monocarbonyls that include formaldehyde (FA), carbon monoxide (CO) and phosgene are discussed. One of the most exciting advances in this review is the summary of fluorescent probes of dicarbonyl compounds.

An efficient PeT based fluorescent probe for mapping mitochondrial oxidative stress produced via the Nox2 pathway.

The human innate immune system eliminates invading pathogens through phagocytosis. The first step of this process is activating the nicotinamide adenine dinucleotide phosphate oxidase (Nox2) that utilizes NADPH to produce superoxide anion radicals and other reactive oxygen species (ROS). These ROS then alter the mitochondrial membrane potential and increase peroxide in the mitochondria. The peroxide reacts with myeloperoxidase (MPO) and chloride ions to produce pro-inflammatory oxidant hypochlorous acid (HOCl), which causes oxidative stress leading to cell death. The adverse effects of HOCl are highly associated with cardiovascular disease, neurodegenerative disorders, acute lung injuries, inflammatory diseases, and cancer. Therefore, mapping HOCl in the Nox2 pathway is crucial for an in-depth understanding of the innate immune system. Herein, we developed a unique pentacyclic pyridinium probe, PM-S, that exhibited efficient photoinduced electron transfer (PeT) with HOCl triggered methyl(phenyl)sulfane. PM-S showed several advantages, including better chemical stability, large Stokes shifts (>6258 cm-1), high sensitivity (∼50 nM) and specificity to mitochondria, compared to its parent pyrylium PY-S derivative. This probe is also efficient in studying the HOCl produced via the Nox2 pathway in HepG2 and HeLa cells. Analysis using a simple microplate reader and FACS analysis with various inhibitors and inducers supported the mechanistic understanding of Nox2, which can offer an advanced platform for monitoring the inflammatory process more efficiently.

IndiFluors: A New Full-Visible Color-Tunable Donor-Acceptor-Donor (D1-A-D2) Fluorophore Family for Ratiometric pH Imaging during Mitophagy.

Full-visible color-tunable new fluorophores are essential in bioimaging research. However, it is significantly challenging to design fluorophores with the desired optical and biological properties owing to their structural complexity. We report a unified design of an interesting molecular framework, IndiFluors, based on the principle of a donor-acceptor-donor (D1-A-D2) system. The IndiFluors comprise pyrylium, pyridinium, and pyridine derivatives, which exhibit full-visible emission color (375-700 nm) by varying donor and acceptor strengths of the core scaffolds. With a minimal change of structure, the bright fluorophores (Φ: 0.96) can be tuned to become nonfluorescent (Φ: 0.01), which is well explained by time-dependent density functional theory (TD-DFT/PCM) by oscillator strengths in the S1 state. Within IndiFluors, pyridinium offers several advantages, including a large Stokes shift (∼154 nm) and excellent stability, compared to pentacyclic pyrylium fluorophores. Especially, the designed probe, PM-Mito-OH, demonstrated specific colocalization in mitochondria and a monitored ratiometric pH change during mitochondrial damage, autolysosomes, and the mitophagy process. Hence, IndiFluors and the derived probe show great potential for cellular pH imaging in live cells while exhibiting minimal cytotoxicity.

ICT based water-soluble fluorescent probe for discriminating mono and dicarbonyl species and analysis in foods.

A unique and highly water-soluble ICT-based fluorescent probe is developed for efficient detection and discrimination of reactive monocarbonyl formaldehyde (FA) from dicarbonyl methylglyoxal (MGO)/glyoxal (GO) by modulating the ICT process, which was confirmed by photophysical and TD-DFT analysis. The probe is applied in cellular imaging and quantifying FA in preserved food and MGO in manuka honey.

A single benzene fluorescent probe for efficient formaldehyde sensing in living cells using glutathione as an amplifier.

Formaldehyde (FA), a simple reactive carbonyl molecule, is endogenously produced in the cell at various physiological condition. At elevated level, FA causes severe cell toxicity as well as damage in macromolecules such proteins and DNA. For detecting FA in living cell, we identify a small but effective fluorescent turn on probe comprising single benzene-based orothophenylenediamine compound. Further study reveals that carboxylic group in orothophenylenediamine plays the important role in enhancing fluorescent signal than another electron withdrawing group. It is even interesting to observe the occurrence of fluorescent enhancement in glutathione (GSH) environment which is generally abundant in every cell. Our probe enables to detect FA over other bio-analytes efficiently with limit of detection of 123 nM and 355-fold of enhancement in cellular mimicking conditions. Moreover, this probe could be useful in discriminating cell that has high concentration of FA as well as GSH.

Highly selective chemosensor for reactive carbonyl species based on simple 1,8-diaminonaphthalene.

Reactive carbonyl species (RCSs) including one carbon formaldehyde (FA) and dicarbonyl compounds such as methylglyoxal (MGO) and glyoxal (GO) are produced during demethylase reactions and various glucose metabolic pathways respectively. Elevation of the RCSs concentrations in cells is due to abnormal DNA damage, glycation adducts with macromolecules that lead to various neurotoxic diseases. Hence, regular monitoring of these RCSs with an easy tool is of utmost interest. However, conventional methods such as chromatography and mass spectrometry for the detection of these species are not so economically viable. These issues were well addressed by the non-invasive reactivity-based fluorescence techniques. However, tedious synthesis, only specific to either mono aldehyde is limited to detect multiple RCSs in physiologies by synthesized fluorophores. An alternative, simple small molecules are widely applied as commercial biomarkers such as terephthalate and 2,3-diaminonaphthalene (NAP) for hydroxy radical (OH·) and nitric oxide (NO) respectively. Herein, we report an analogue of NAP, 1,8-diamino naphthalene (DAN) is an efficient chemosensor for highly sensitive detection of FA, MGO and GO with minimum detection limits of 0.95-3.97 µM. Surprisingly, DAN shows a "turn on" response towards RCSs but remaining silent towards NO which are exactly opposite to commercial probe NAP. Exogenous RCSs imaging in vitro cancerous cells shows the efficacy of the probe and its potential application for RCSs monitoring in cancer cells, generation of toxic byproducts.

Quinone-Based Antitumor Agent Sepantronium Bromide (YM155) Causes Oxygen-Independent Redox-Activated Oxidative DNA Damage.

Sepantronium bromide (YM155) is a small molecule antitumor agent currently in phase II clinical trials. Although developed as survivin suppressor, YM155's primary mode of action has recently been found to be DNA damage. However, the mechanism of DNA damage by YM155 is still unknown. Knowing the mechanism of action of an anticancer drug is necessary to formulate a rational drug combination and select a cancer type for achieving maximum clinical efficacy. Using cell-based assays, we showed that YM155 causes extensive DNA cleavage and reactive oxygen species generation. DNA cleavage by YM155 was found to be inhibited by radical scavengers and desferal. The reducing agent DTT and the cellular reducing system xanthine/xanthine oxidase were found to reductively activate YM155 and cause DNA cleavage. Unlike quinones, DNA cleavage by YM155 occurs in the presence of catalase and under hypoxic conditions, indicating that hydrogen peroxide and oxygen are not necessary. Although YM155 is a quinone, it does not follow a typical quinone mechanism. Consistent with these observations, a mechanism has been proposed that suggests that YM155 can cause oxidative DNA cleavage upon 2-electron reductive activation.