Development of a solid-supported light-triggered nitric oxide donor.

Nitric Oxide (NO) photocleavable donors are useful tools for interrogating nitric oxide signalling and have potential use in photopharmacological applications. There is currently intensive research into newer methods to improve NO release and kinetic profiles. Herein, we report the design and synthesis of a solid-supported photocleavable NO donor synthesized by ligating an N-nitroso photocleavable nitric oxide derivative to a TentaGel® polymer resin bead. Illumination with 365 nm light released nitric oxide that could be tracked via a turn-on fluorescence response (λex = 450 nm, λem = 545 nm) and measured using the Griess assay and diaminorhodamine derivatives. These beads were further shown to be compatible with living A549 cells and had the ability to deliver greater concentrations of nitric oxide to cells proximal to a bead versus cells at more distal locations within the same well.

Kinetics-Based Quantification of Peroxynitrite Using the Oxidative Decarbonylation of Isatin.

Peroxynitrite and its radical decomposition products are highly reactive nitrogen and oxygen species that can influence the balance between health and disease in multiple organ systems. Despite vigorous research activity, real-time quantitative monitoring of peroxynitrite generated by donor compounds remains challenging. Here, we report a kinetics-based fluorescence method for quantitative tracking of peroxynitrite generation using the oxidative decarbonylation of isatin to form anthranilic acid as a fluorescent probe. This method relies on knowledge of the rate of the reaction of peroxynitrite with the probe, which we measure using stopped-flow fluorescence techniques. To the best of our knowledge, this is the first optical method capable of providing real-time quantitative measures of peroxynitrite concentrations generated from donor compounds, as demonstrated herein for SIN-1 and Angeli's salt.

Seeking Illumination: The Path to Chemiluminescent 1,2-Dioxetanes for Quantitative Measurements and In Vivo Imaging.

Chemiluminescence is a fascinating phenomenon that evolved in nature and has been harnessed by chemists in diverse ways to improve life. This Account tells the story of our research group's efforts to formulate and manifest spiroadamantane 1,2-dioxetanes with triggerable chemiluminescence for imaging and monitoring important reactive analytes in living cells, animals, and human clinical samples. Analytes like reactive sulfur, oxygen and nitrogen species, as well as pH and hypoxia can be indicators of cellular function or dysfunction and are often implicated in the causes and effects of disease. We begin with a foundation in binding-based and activity-based fluorescence imaging that has provided transformative tools for understanding biological systems. The intense light sources required for fluorescence excitation, however, introduce autofluorescence and light scattering that reduces sensitivity and complicates in vivo imaging. Our work and the work of our collaborators were the first to demonstrate that spiroadamantane 1,2-dioxetanes had sufficient brightness and biological compatibility for in vivo imaging of enzyme activity and reactive analytes like hydrogen sulfide (H2S) inside of living mice. This launched an era of renewed interest in 1,2-dioxetanes that has resulted in a plethora of new chemiluminescence imaging agents developed by groups around the world. Our own research group focused its efforts on reactive sulfur, oxygen, and nitrogen species, pH, and hypoxia, resulting in a large family of bright chemiluminescent 1,2-dioxetanes validated for cell monitoring and in vivo imaging. These chemiluminescent probes feature low background and high sensitivity that have been proven quite useful for studying signaling, for example, the generation of peroxynitrite (ONOO-) in cellular models of immune function and phagocytosis. This high sensitivity has also enabled real-time quantitative reporting of oxygen-dependent enzyme activity and hypoxia in living cells and tumor xenograft models. We reported some of the first ratiometric chemiluminescent 1,2-dioxetane systems for imaging pH and have introduced a powerful kinetics-based approach for quantification of reactive species like azanone (nitroxyl, HNO) and enzyme activity in living cells. These tools have been applied to untangle complex signaling pathways of peroxynitrite production in radiation therapy and as substrates in a split esterase system to provide an enzyme/substrate pair to rival luciferase/luciferin. Furthermore, we have pushed chemiluminescence toward commercialization and clinical translation by demonstrating the ability to monitor airway hydrogen peroxide in the exhaled breath of asthma patients using transiently produced chemiluminescent 1,2-dioxetanedione intermediates. This body of work shows the powerful possibilities that can emerge when working at the interface of light and chemistry, and we hope that it will inspire future scientists to seek out ever brighter and more illuminating ideas.

Energy Transfer Chemiluminescent Spiroadamantane 1,2-Dioxetane Probes for Bioanalyte Detection and Imaging.

Chemiluminescence imaging of bioanalytes using spiroadamantane 1,2-dioxetanes has gained significant attention due to improved signal-to-noise ratios and imaging depth compared to excitation-based probes, as well as their modifiable scaffolds that offer analyte-specific responses and tunable emissive properties. Among several strategies employed to amplify signals under aqueous conditions and to shift the emission into the bio-relevant red region, energy transfer to an adjacent fluorophore is a popular and effective method. This Minireview highlights spiroadamantane 1,2-dioxetane-based probes that operate via an energy transfer mechanism to detect bioanalytes both in vitro and in vivo. Probes that display both non-covalent and covalent interactions with fluorophores, as well as their applications in imaging specific analytes will be discussed.

Chemiluminescent 1,2-Dioxetane Iridium Complexes for Near-Infrared Oxygen Sensing.

Chemiluminescent iridium-based sensors which demonstrate oxygen dependent responses have been developed. The molecular probes, named IrCL-1, IrCL-2 and IrCL-3 consist of oxygen-sensitive iridium complexes attached to a spiroadamantane 1,2 dioxetane and operate via energy transfer from the chemiexcited benzoate to the corresponding iridium(III) complex. Complexing the iridium(III) center with π-extended ligands results in emission in the biologically relevant, near-infrared (NIR) region. All probes demonstrate varying oxygen tolerance, with IrCL-1 being the most oxygen sensitive. These probes have been further utilized for in vitro ratiometric imaging of oxygen, as well as for intraperitoneal, intramuscular and intratumoral imaging in live mice. To our knowledge, these are the first iridium-based chemiluminescent probes that have been employed for in vitro ratiometric oxygen sensing, and for in vivo tumor imaging.

Chemiluminescent Measurement of Hydrogen Peroxide in the Exhaled Breath Condensate of Healthy and Asthmatic Adults.

Reactive oxygen species are centrally involved in the pathophysiology of airway diseases such as asthma and chronic obstructive pulmonary disease. This study reports the development of a chemiluminescence assay and a device for measuring hydrogen peroxide in the exhaled breath condensate of asthma patients and healthy participants. A stand-alone photon detection device was constructed for use with an optimized chemiluminescence assay. Calibrations using a catalase control to scavenge residual hydrogen peroxide in calibrant solutions provided analytically sensitive and specific measurements. We evaluated exhaled breath condensate hydrogen peroxide in 60 patients (ages 20-83; 30 healthy patients and 30 asthma patients) recruited from the John Peter Smith Hospital Network. The exhaled breath condensate hydrogen peroxide concentrations trended toward higher values in asthma patients compared to healthy participants (mean 142.5 vs 115.5 nM; p = 0.32). Asthma patients who had not used an albuterol rescue inhaler in the past week were compared to those who had and showed a trend toward higher hydrogen peroxide levels (mean 172.8 vs 115.9 nM; p = 0.25), and these patients also trended toward higher hydrogen peroxide than healthy participants (mean 172.8 vs 115.5 nM; p = 0.14). This pilot study demonstrates the ability of the newly developed assay and device to measure exhaled breath condensate hydrogen peroxide in asthma patients and healthy participants. The trends observed in this study are in agreement with previous literature and warrant further investigation of using this system to measure exhaled breath condensate hydrogen peroxide for monitoring oxidative stress in asthma.

A Chemiluminescent Probe for HNO Quantification and Real-Time Monitoring in Living Cells.

Azanone (HNO) is a reactive nitrogen species with pronounced biological activity and high therapeutic potential for cardiovascular dysfunction. A critical barrier to understanding the biology of HNO and furthering clinical development is the quantification and real-time monitoring of its delivery in living systems. Herein, we describe the design and synthesis of the first chemiluminescent probe for HNO, HNOCL-1, which can detect HNO generated from concentrations of Angeli's salt as low as 138 nm with high selectivity based on the reaction with a phosphine group to form a self-cleavable azaylide intermediate. We have capitalized on this high sensitivity to develop a generalizable kinetics-based approach, which provides real-time quantitative measurements of HNO concentration at the picomolar level. HNOCL-1 can monitor dynamics of HNO delivery in living cells and tissues, demonstrating the versatility of this method for tracking HNO in living systems.

Characterization and Biological Activity of a Hydrogen Sulfide-Releasing Red Light-Activated Ruthenium(II) Complex.

Hydrogen sulfide (H2S) is a biological gasotransmitter that has been employed for the treatment of ischemia-reperfusion injury. Despite its therapeutic value, the implementation of this gaseous molecule for this purpose has required H2S-releasing prodrugs for effective intracellular delivery. The majority of these prodrugs, however, spontaneously release H2S via uncontrolled hydrolysis. Here, we describe a Ru(II)-based H2S-releasing agent that can be activated selectively by red light irradiation. This compound operates in living cells, increasing intracellular H2S concentration only upon irradiation with red light. Furthermore, the red light irradiation of this compound protects H9c2 cardiomyoblasts from an in vitro model of ischemia-reperfusion injury. These results validate the use of red light-activated H2S-releasing agents as valuable tools for studying the biology and therapeutic utility of this gasotransmitter.

Energy transfer chemiluminescence for ratiometric pH imaging.

Chemiluminescence imaging offers a low background and high sensitivity approach to imaging analytes in living cells and animals. Intensity-based measurements have been developed, but require careful consideration of kinetics, probe localization, and fluctuations in quantum yield, all of which complicate quantification. Here, we report a ratiometric strategy for quantitative chemiluminescence imaging of pH. The strategy relies on an energy transfer cascade of chemiluminescence emission from a spiroadamantane 1,2-dioxetane to a ratiometric pH indicator via fluorescent dyes in Enhancer solutions. Monitoring the pH-dependent changes in chemiluminescence emission at multiple wavelengths enables ratiometric imaging and quantification of pH independent from variations due to kinetics and probe concentration.

Ultrasensitive Chemiluminescent Detection of Cathepsin†B: Insights into the New Frontier of Chemiluminescent Imaging.

Let there be light: Chemiluminescence provides a bright detection signal against a dark background and offers an excellent signal-to-noise ratio for analysis. Now, a chemiluminescent probe for cathepsin B has been developed that provides a 16,000-fold improvement in sensitivity for detecting protease activity.

A chemiluminescent platform for smartphone monitoring of H2O2 in human exhaled breath condensates.

Noninvasive measurement of oxidative markers in clinical samples has the potential to rapidly provide information for disease management, but is limited by the need for expensive analytical instrumentation that precludes home monitoring or point-of-care applications. We have developed a simple to use diagnostic platform for airway hydrogen peroxide (H2O2) that combines optimized reaction-based chemiluminescent designs with an inexpensive home-built darkbox and readily available smartphone cameras. Specialized photography software applications and analysis of pixel intensity enables quantification of sample concentrations. Using this platform, sample H2O2 concentrations as low as 264nM can be detected. The platform has been used to measure H2O2 in the exhaled breath condensates of human subjects, showing good agreement with the standard Amplex Red assay.

In Vivo Chemiluminescent Imaging Agents for Nitroreductase and Tissue Oxygenation.

Tissue oxygenation is a driving parameter of the tumor microenvironment, and hypoxia can be a prognostic indicator of aggressiveness, metastasis, and poor response to therapy. Here, we report a chemiluminescence imaging (CLI) agent based on the oxygen-dependent reduction of a nitroaromatic spiroadamantane 1,2-dioxetane scaffold. Hypoxia ChemiLuminescent Probe 2 (HyCL-2) responds to nitroreductase with ∼170-fold increase in luminescence intensity and high selectivity for enzymatic reductase versus other small molecule reductants. HyCL-2 can image exogenous nitroreductase in vitro and in vivo in living mice, and total luminescent intensity is increased by ∼5-fold under low oxygen conditions. HyCL-2 is demonstrated to report on tumor oxygenation during an oxygen challenge in H1299 lung tumor xenografts grown in a murine model as independently confirmed using multispectral optoacoustic tomography (MSOT) imaging of hemoglobin oxygenation.

Cell-trappable fluorescent probes for endogenous hydrogen sulfide signaling and imaging H2O2-dependent H2S production.

Hydrogen sulfide (H2S) is a reactive small molecule generated in the body that can be beneficial or toxic owing to its potent redox activity. In living systems, disentangling the pathways responsible for H2S production and their physiological and pathological consequences remains a challenge in part due to a lack of methods for monitoring changes in endogenous H2S fluxes. The development of fluorescent probes with appropriate selectivity and sensitivity for monitoring production of H2S at biologically relevant signaling levels offers opportunities to explore its roles in a variety of systems. Here we report the design, synthesis, and application of a family of azide-based fluorescent H2S indicators, Sulfidefluor-4, Sulfidefluor-5 acetoxymethyl ester, and Sulfidefluor-7 acetoxymethyl ester, which offer the unique capability to image H2S generated at physiological signaling levels. These probes are optimized for cellular imaging and feature enhanced sensitivity and cellular retention compared with our previously reported molecules. In particular, Sulfidefluor-7 acetoxymethyl ester allows for direct, real-time visualization of endogenous H2S produced in live human umbilical vein endothelial cells upon stimulation with vascular endothelial growth factor (VEGF). Moreover, we show that H2S production is dependent on NADPH oxidase-derived hydrogen peroxide (H2O2), which attenuates VEGF receptor 2 phosphorylation and establishes a link for H2S/H2O2 crosstalk.

Ylide mediated carbonyl homologations for the preparation of isatin derivatives.

An exceptionally mild method for the preparation of isatin derivatives has been developed using a sulfur ylide mediated carbonyl homologation sequence starting from anthranilic acid precursors. This method proceeds at ambient temperature via a sulfur ylide intermediate without the need for protection of the amine or chromatographic isolation of the intermediate ylide. Gentle oxidation of the sulfur ylides provides isatin derivatives with N-H, N-alkyl, N-aryl substitution, electron-rich and electron-poor aromatic rings, and heterocyclic aromatic systems. We anticipate that this method will greatly expand the accessibility of complex isatin derivatives.

Study of non-spherical bubble oscillations near a surface in a weak acoustic standing wave field.

The interaction of acoustically driven bubbles with a wall is important in many applications of ultrasound and cavitation, as the close boundary can severely alter the bubble dynamics. In this paper, the non-spherical surface oscillations of bubbles near a surface in a weak acoustic standing wave field are investigated experimentally and numerically. The translation, the volume, and surface mode oscillations of bubbles near a flat glass surface were observed by a high speed camera in a standing wave cell at 46.8 kHz. The model approach is based on a modified Keller-Miksis equation coupled to surface mode amplitude equations in the first order, and to the translation equations. Modifications are introduced due to the adjacent wall. It was found that a bubble's oscillation mode can change in the presence of the wall, as compared to the bubble in the bulk liquid. In particular, the wall shifts the instability pressure thresholds to smaller driving frequencies for fixed bubble equilibrium radii, or to smaller equilibrium radii for fixed excitation frequency. This can destabilize otherwise spherical bubbles, or stabilize bubbles undergoing surface oscillations in the bulk. The bubble dynamics observed in experiment demonstrated the same trend as the theoretical results.

Exploring the Structural Space of Chemiluminescent 1,2-Dioxetanes.

Chemiluminescent molecules which emit light in response to a chemical reaction are powerful tools for the detection and measurement of biological analytes and enable the understanding of complex biochemical processes in living systems. Triggerable chemiluminescent 1,2-dioxetanes have been studied and tuned over the past decades to advance quantitative measurement of biological analytes and molecular imaging in live cells and animals. A crucial determinant of success for these 1,2-dioxetane based sensors is their chemical structure, which can be manipulated to achieve desired chemical properties. In this Perspective, we survey the structural space of triggerable 1,2-dioxetane and assess how their design features affect chemiluminescence properties including quantum yield, emission wavelength, and decomposition kinetics. Based on this appraisal, we identify some structural modifications of 1,2-dioxetanes that are ripe for exploration in the context of chemiluminescent biological sensors.

Oxygen-Sensing Chemiluminescent Iridium(III) 1,2-Dioxetanes: Unusual Coordination and Activity.

Next generation chemiluminescent iridium 1,2-dioxetane complexes have been developed which consist of the Schaap's 1,2-dioxetane scaffold directly attached to the metal center. This was achieved by synthetically modifying the scaffold precursor with a phenylpyridine moiety, which can act as a ligand. Reaction of this scaffold ligand with the iridium dimer [Ir(BTP)2(µ-Cl)]2 (BTP = 2-(benzo[b]thiophen-2-yl)pyridine) yielded isomers which depict ligation through either the cyclometalating carbon or, interestingly, the sulfur atom of one BTP ligand. Their corresponding 1,2-dioxetanes display chemiluminescent responses in buffered solutions, exhibiting a single, red-shifted peak at 600 nm. This triplet emission was effectively quenched by oxygen, yielding in vitro Stern-Volmer constants of 0.1 and 0.009 mbar-1 for the carbon-bound and sulfur compound, respectively. Lastly, the sulfur-bound dioxetane was further utilized for oxygen sensing in muscle tissue of living mice and xenograft models of tumor hypoxia, depicting the ability of the probe chemiluminescence to penetrate biological tissue (total flux ~ 106 p/s).

A reaction-based fluorescent probe for selective imaging of carbon monoxide in living cells using a palladium-mediated carbonylation.

Carbon monoxide is a member of the gasotransmitter family, which also includes NO and H(2)S, and has been implicated in a variety of pathological and physiological conditions. Whereas exogenous therapeutic additions of CO to tissues and whole animals have been well-studied, the real-time spatial and temporal tracking of CO at the cellular level remains an open challenge. Here we report a new type of turn-on fluorescent probe for selective CO detection based on palladium-mediated carbonylation reactivity. CO Probe 1 (COP-1) is capable of detecting CO both in aqueous buffer and in live cells with high selectivity over a range of biologically relevant reactive small molecules, providing a potentially powerful approach for interrogating its chemistry in biological systems.

Boronate oxidation as a bioorthogonal reaction approach for studying the chemistry of hydrogen peroxide in living systems.

Reactive oxygen species (ROS), such as hydrogen peroxide, are important products of oxygen metabolism that, when misregulated, can accumulate and cause oxidative stress inside cells. Accordingly, organisms have evolved molecular systems, including antioxidant metalloenzymes (such as superoxide dismutase and catalase) and an array of thiol-based redox couples, to neutralize this threat to the cell when it occurs. On the other hand, emerging evidence shows that the controlled generation of ROS, particularly H(2)O(2), is necessary to maintain cellular fitness. The identification of NADPH oxidase enzymes, which generate specific ROS and reside in virtually all cell types throughout the body, is a prime example. Indeed, a growing body of work shows that H(2)O(2) and other ROS have essential functions in healthy physiological signaling pathways. The signal-stress dichotomy of H(2)O(2) serves as a source of motivation for disentangling its beneficial from its detrimental effects on living systems. Molecular imaging of this oxygen metabolite with reaction-based probes is a powerful approach for real-time, noninvasive monitoring of H(2)O(2) chemistry in biological specimens, but two key challenges to studying H(2)O(2) in this way are chemoselectivity and bioorthogonality of probe molecules. Chemoselectivity is problematic because traditional methods for ROS detection suffer from nonspecific reactivity with other ROS. Moreover, some methods require enzymatic additives not compatible with live-cell or live-animal specimens. Additionally, bioorthogonality requires that the reactions must not compete with or disturb intrinsic cellular chemistry; this requirement is particularly critical with thiol- or metal-based couples mediating the major redox events within the cell. Chemoselective bioorthogonal reactions, such as alkyne-azide cycloadditions and related click reactions, the Staudinger-Bertozzi ligation, and the transformations used in various reaction-based molecular probes, have found widespread application in the modification, labeling, and detection of biological molecules and processes. In this Account, we summarize H(2)O(2) studies from our laboratory using the H(2)O(2)-mediated oxidation of aryl boronates to phenols as a bioorthogonal approach to detect fluxes of this important ROS in living systems. We have installed this versatile switch onto organic and inorganic scaffolds to serve as "turn-on" probes for visible and near-infrared (NIR) fluorescence, ratiometric fluorescence, time-gated lanthanide luminescence, and in vivo bioluminescence detection of H(2)O(2) in living cells and animals. Further chemical and genetic manipulations target these probes to specific organelles and other subcellular locales and can also allow them to be trapped intracellularly, enhancing their sensitivity. These novel chemical tools have revealed fundamental new biological insights into the production, localization, trafficking, and in vivo roles of H(2)O(2) in a wide variety of living systems, including immune, cancer, stem, and neural cell models.