Oxidative stress is a common feature shared by many diseases, including neurodegenerative diseases. Factors that contribute to cellular oxidative stress include elevated levels of reactive oxygen species, diminished availability of detoxifying thiols, and the misregulation of metal ions (both redox-active iron and copper as well as non-redox active calcium and zinc). Deciphering how each of these components interacts to contribute to oxidative stress presents an interesting challenge. Fluorescent sensors can be powerful tools for detecting specific analytes within a complicated cellular environment. Reviewed here are several classes of small molecule fluorescent sensors designed to detect several molecular participants of oxidative stress. We focus our review on describing the design, function and application of probes to detect metal cations, reactive oxygen species, and intracellular thiol-containing compounds. In addition, we highlight the intricacies and complications that are often faced in sensor design and implementation.

A novel platinum(II) photocaged complex called [Pt(cage)] has been prepared and characterized by X-ray crystallography. The complex contains a photolabile nitrophenyl group incorporated into the backbone of a tetradentate ligand that contains two pyridyl and two amide nitrogen donor sites. The intact complex is unreactive toward ligand-exchange reactions until activation with UV light cleaves the ligand backbone, releasing a Pt II complex that more readily exchanges

A fluorescent sensor prochelator, FlamB (fluorescein hydrizido 2-imidophenylboronic ester), has been developed that selectively probes for copper under conditions of oxidative stress. High levels of hydrogen peroxide trigger release of a boronic ester masking group from the prochelator to unveil a metal chelator, FlamS (fluorescein hydrizido 2-imidophenol), that provides a modest fluorescence increase in response to Cu2+ but not other metal ions. X-ray crystal structures of FlamB, FlamS, and Cu-bound FlamS are all reported. The fluorescence turn-on results from opening of a fluorescein spirolactam ring upon Cu2+ binding to FlamS in aqueous solution. Oxidation of the aryl boronic ester of FlamB to the metal-binding phenol of FlamS proceeds in organic solvents. However, in aqueous solution a competing mechanism occurs due to hydrolytic instability of the masked prochelator. Hydrolysis of FlamB leads to formation of fluorescein hydrazide, which interacts with copper or H2O2 to produce fluorescein and a significant fluorescence increase.

A new type of Cu 2+ fluorescent sensor, coucage, has been prepared with a photosensitive nitrophenyl group incorporated into the backbone of a coumarin-tagged tetradentate ligand. Coucage provides a selective fluorescence response for Cu 2+ over other biologically relevant metal ions. Coordination of Cu 2+ dims the fluorescence output until irradiation with UV light cleaves the ligand backbone, which relieves the copper-induced quenching to provide a turn-on response. Experiments in live MCF-7 cells show that coucage can be used for detecting changes in intracellular Cu 2+ upon the addition of excess exogenous copper. If improvements can be made to increase its affinity for copper, this new type of turn-on sensor could be used as a tool for visualizing the cellular distribution of labile copper to gain insight into the mechanisms of copper trafficking.Copper, the third most abundant transition metal in the human body, plays a critical role in many fundamental physiological processes; however, it also catalyzes the production of highly reactive oxygen species that damage biomolecules.1 Due to copper's dual nature, cells have developed strict regulatory processes to control its cellular distribution.1 Alterations in copper homeostasis are linked to neurodegenerative diseases such as Menkes and Wilson diseases, Alzheimer's, familial amyotrophic lateral sclerosis, and prion diseases. 2 Being able to visualize the cellular distribution of copper in both its physiological oxidation states, Cu + and Cu 2+ , would offer insight into how cells acquire, maintain, and utilize copper while suppressing its toxicity. Whereas reliable fluorescence sensors exist for Cu + , there are fewer options for detecting Cu 2+ in living cells.3 A common strategy in designing fluorescent probes for metal ions is to link a ligand to a fluorophore such that metal binding causes an increase in fluorescence only in response to the target ion. Cell permeable fluorescent sensors have proven useful for investigating intracellular metal ion distribution, particularly for Ca 2+ ,4 Zn 2+, 5 and Cu + .6 The development of this type of "turn-on" sensor for Cu 2+ , however, is hampered by the fluorescence quenching effect of this paramagnetic metal ion. As a consequence, many Cu 2+ sensors have a "turn-off" mechanism,7 which is generally less sensitive, gives false-positive results, and offers limited spatial resolution. Several examples of turn-on sensors have appeared recently,3 , 8 but limitations include sensing mechanisms that operate only in organic solvent or at non-physiological pH,8a-d low quantum yields in aqueous solution,8e or potential off-target responses.8f-i Therefore, there is a need to develop new strategies that provide a fluorescent turn-on response in order to investigate intracellular Cu 2+ . We present here coucage, a new type of fluorescent sensor that uses UV light to uncage a Cu 2+ -dependent fluorescence response.Katherine.franz@duke.edu. Supporting Information Available: Full experimental details, including synthesis...

Described here is the development of two boronic ester-based fluorescent prochelators, FloB (2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-4(5)-[2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-benzylidene-hydrazinocarbonyl]-benzoic acid) and FloB-SI (2-(6-hydroxy-3-oxo-3Hxanthen-9-yl)-4(5)-[2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyloxy)-benzylidene-hydrazinocarbonyl]-benzoic acid) that show a fluorescence response to a variety of transition metal ions only after reaction with H2O2. Both prochelators’ boronic ester masks are oxidized by H2O2 to reveal a fluorescein-tagged metal chelator, FloS (4(5)-(2-hydroxy-benzylidenehydrazinocarbonyl)-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-benzoic acid). Chelation of Fe3+ or Cu2+ elicits a 70% decrease in the emission signal of FloS, while Zn2+, Ni2+, and Co2+ produce a more modest fluorescence decrease. The conversion of FloB to FloS proceeds in organic solvents, but hydrolytic decomposition of its hydrazone backbone is observed in aqueous solution. However, FloB-SI oxidizes cleanly with H2O2 within 1 h in aqueous solutions to produce FloS. Fluorescence microscopy studies in HeLa cells with FloB-SI show that the sensor’s fluorescence intensity remains unchanged until incubation with exogenous H2O2, which results in a decreased fluorescent signal. Incubation with a competitive chelator restores the emission response, thus suggesting that FloB-SI can effectively report on a H2O2-induced increase in intracellular labilized metal.