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.

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).

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.

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.

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 spiroadamantane-1,2-dioxetanes: Recent advances in molecular imaging and biomarker detection.

Triggered chemiluminescence emission of spiroadamantane-1,2-dioxetanes to detect bioanalytes has fueled the emerging popularity of chemiluminescence imaging in live animals and cells. Recently, a structural evolution of the dioxetane scaffolds towards near-infrared emitters has been observed, and efforts have been made for quantitative and semi-quantitative detection of a wide range of analytes. In this review, we summarize the current chemiluminescence imaging developments of spiroadamantane-1,2-dioxetanes. Specifically, we look at examples which depict whole animal or cellular chemiluminescence imaging of small molecules and enzymes, as well as those that portray their potential diagnostic and therapeutic abilities, with an emphasis on analyte quantification and experimental parameters.

Dark Dynamic Therapy: Photosensitization without Light Excitation Using Chemiluminescence Resonance Energy Transfer in a Dioxetane-Erythrosin B Conjugate.

Reactive oxygen species (e.g., singlet oxygen) are the primary cytotoxic agents used in the clinically approved technique photodynamic therapy (PDT). Although singlet oxygen has high potential to effectively kill tumor cells, its production via light excitation of a photosensitizer has been limited by the penetration depth and delivery of light in tissue. To produce singlet oxygen without light excitation, we describe the use of Schaap's chemiluminescent scaffold comprising an adamantylidene-dioxetane motif. Functionalizing this scaffold with a photosensitizer, Erythrosin B, resulted in spontaneous chemiluminescence resonance energy transfer (CRET) leading to the production of singlet oxygen. We show that this compound is cell permeable and that the singlet oxygen produced via CRET is remarkably efficient in killing cancer cells at low micromolar concentrations. Moreover, we demonstrate that protection of the phenol on the chemiluminescent scaffold with a nitroreductase-responsive trigger group allows for cancer-selective dark dynamic cell death. Here, we present the concept of dark dynamic therapy using a small cell-permeable molecule capable of producing the effects of PDT in cells, without light.

Visible Light Chemical Micropatterning Using a Digital Light Processing Fluorescence Microscope.

Patterning chemical reactivity with a high spatiotemporal resolution and chemical versatility is critically important for advancing revolutionary emergent technologies, including nanorobotics, bioprinting, and photopharmacology. Current methods are complex and costly, necessitating novel techniques that are easy to use and compatible with a wide range of chemical functionalities. This study reports the development of a digital light processing (DLP) fluorescence microscope that enables the structuring of visible light (465-625 nm) for high-resolution photochemical patterning and simultaneous fluorescence imaging of patterned samples. A range of visible-light-driven photochemical systems, including thiol-ene photoclick reactions, Wolff rearrangements of diazoketones, and photopolymerizations, are shown to be compatible with this system. Patterning the chemical functionality onto microscopic polymer beads and films is accomplished with photographic quality and resolutions as high as 2.1 µm for Wolff rearrangement chemistry and 5 µm for thiol-ene chemistry. Photoactivation of molecules in living cells is demonstrated with single-cell resolution, and microscale 3D printing is achieved using a polymer resin with a 20 µm xy-resolution and a 100 µm z-resolution. Altogether, this work debuts a powerful and easy-to-use platform that will facilitate next-generation nanorobotic, 3D printing, and metamaterial technologies.

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.

Defects Formation and Amorphization of Zn-MOF-74 Crystals by Post-Synthetic Interactions with Bidentate Adsorbates.

The controlled introduction of defects into MOFs is a powerful strategy to induce new physiochemical properties and improve their performance for target applications. Herein, we present a new strategy for defect formation and amorphization of the canonical MOF-74 frameworks based on fine-tuning of adsorbate-framework interactions in the metal congener, hence introducing structural defects. Specifically, we demonstrate that controlled interactions between the MOF and bidentate ligands adsorbed in the pores initiates defect formation and eventual amorphization of the crystal. These structural features unlock properties that are otherwise absent in the ordered framework, such as broad-band fluorescence. The ability to introduce defects by adsorbate-framework interactions, coupled with the inherent tunability and modularity of these structures, provides a new route for the synthesis of diverse heterogeneous and hybrid materials.

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.

Imaging Mitochondrial Hydrogen Peroxide in Living Cells.

Hydrogen peroxide (H2O2) produced from mitochondria is intimately involved in human health and disease, but is challenging to selectively monitor inside living systems. The fluorescent probe MitoPY1 provides a practical tool for imaging mitochondrial H2O2 and has been demonstrated to function in a variety of diverse cell types. In this chapter, we describe the synthetic preparation of the small molecule probe MitoPY1 , methods for validating this probe in vitro and in live cells, and an example procedure for measuring mitochondrial H2O2 in a cell culture model of Parkinson's disease.

Boosting nitric oxide in stress and respiratory infection: Potential relevance for asthma and COVID-19.

Nitric oxide (NO) is a ubiquitous signaling molecule that is critical for supporting a plethora of processes in biological organisms. Among these, its role in the innate immune system as a first line of defense against pathogens has received less attention. In asthma, levels of exhaled NO have been utilized as a window into airway inflammation caused by allergic processes. However, respiratory infections count among the most important triggers of disease exacerbations. Among the multitude of factors that affect NO levels are psychological processes. In particular, longer lasting states of psychological stress and depression have been shown to attenuate NO production. The novel SARS-CoV-2 virus, which has caused a pandemic, and with that, sustained levels of psychological stress globally, also adversely affects NO signaling. We review evidence on the role of NO in respiratory infection, including COVID-19, and stress, and argue that boosting NO bioavailability may be beneficial in protection from infections, thus benefitting individuals who suffer from stress in asthma or SARS-CoV-2 infection.

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.

Ratiometric pH Imaging Using a 1,2-Dioxetane Chemiluminescence Resonance Energy Transfer Sensor in Live Animals.

Regulation of physiological pH is integral for proper whole body and cellular function, and disruptions in pH homeostasis can be both a cause and effect of disease. In light of this, many methods have been developed to monitor pH in cells and animals. In this study, we report a chemiluminescence resonance energy transfer (CRET) probe Ratio-pHCL-1, composed of an acrylamide 1,2-dioxetane chemiluminescent scaffold with an appended pH-sensitive carbofluorescein fluorophore. The probe provides an accurate measurement of pH between 6.8 and 8.4, making it a viable tool for measuring pH in biological systems. Further, its ratiometric output is independent of confounding variables. Quantification of pH can be accomplished using both common luminescence spectroscopy and advanced optical imaging methods. Using an IVIS Spectrum, pH can be measured through tissue with Ratio-pHCL-1, which is shown in vitro and calibrated in sacrificed mouse models. Intraperitoneal injections of Ratio-pHCL-1 into live mice show high photon outputs and consistent increases in the flux ratio when measured at pH 6, 7, and 8.

Development of a Split Esterase for Protein-Protein Interaction-Dependent Small-Molecule Activation.

Split reporters based on fluorescent proteins and luciferases have emerged as valuable tools for measuring interactions in biological systems. Relatedly, biosensors that transduce measured input signals into outputs that influence the host system are key components of engineered gene circuits for synthetic biology applications. While small-molecule-based imaging agents are widely used in biological studies, and small-molecule-based drugs and chemical probes can target a range of biological processes, a general method for generating a target small molecule in a biological system based on a measured input signal is lacking. Here, we develop a proximity-dependent split esterase that selectively unmasks ester-protected small molecules in an interaction-dependent manner. Exploiting the versatility of an ester-protected small-molecule output, we demonstrate fluorescent, chemiluminescent, and pharmacological probe generation, each created by masking key alcohol functional groups on a target small molecule. We show that the split esterase system can be used in combination with ester-masked fluorescent or luminescent probes to measure protein-protein interactions and protein-protein interaction inhibitor engagement. We demonstrate that the esterase-based reporter system is compatible with other commonly used split reporter imaging systems for the simultaneous detection of multiple protein-protein interactions. Finally, we develop a system for selective small-molecule-dependent cell killing by unmasking a cytotoxic molecule using an inducible split esterase. Presaging utility in future synthetic biology-based therapeutic applications, we also show that the system can be used for intercellular cell killing via a bystander effect, where one activated cell unmasks a cytotoxic molecule and kills cells physically adjacent to the activated cells. Collectively, this work illustrates that the split esterase system is a valuable new addition to the split protein toolbox, with particularly exciting potential in synthetic biology applications.

Optimizing Targeted Inhibitors of P-Glycoprotein Using Computational and Structure-Guided Approaches.

Overexpression of ABC transporters like P-glycoprotein (P-gp) has been correlated with resistances in cancer chemotherapy. Intensive efforts to identify P-gp inhibitors for use in combination therapy have not led to clinically approved inhibitors to date. Here, we describe computational approaches combined with structure-based design to improve the characteristics of a P-gp inhibitor previously identified by us. This hit compound represents a novel class of P-gp inhibitors that specifically targets and inhibits P-gp ATP hydrolysis while not being transported by the pump. We describe here a new program for virtual chemical synthesis and computational assessment, ChemGen, to produce hit compound variants with improved binding characteristics. The chemical syntheses of several variants, efficacy in reversing multidrug resistance in cell culture, and biochemical assessment of the inhibition mechanism are described. The usefulness of the computational predictions of binding characteristics of the inhibitor variants is discussed and compared to more traditional structure-based approaches.

Kinetics-Based Measurement of Hypoxia in Living Cells and Animals Using an Acetoxymethyl Ester Chemiluminescent Probe.

Oxygenation and tissue hypoxia play critical roles in mammalian biology and contribute to aggressive phenotypes in cancerous tumors, driving research to develop accurate and easy-to-implement methods for monitoring hypoxia in living cells and animal models. This study reports the chemiluminescent probe HyCL-4-AM, which contains a nitroaromatic sensing moiety and, importantly, an acetoxymethyl (AM) ester that dramatically improves operation in cells and animals. HyCL-4-AM provides a selective 60 000-fold increase in luminescence emission in the presence of rat liver microsomes (RLM). For cellular operation, the chemiluminescence response kinetics is sharply dependent on oxygen levels, enabling highly significant and reproducible measurement of hypoxia in living cells. Whole animal imaging experiments in muscle tissue and tumor xenografts show that HyCL-4-AM can differentiate between well oxygenated muscle tissue and hypoxic tumors, demonstrating potential for monitoring tumor reoxygenation via hyperoxic treatment.