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).

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.

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.

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.

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.

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.

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.

Azide-based fluorescent probes: imaging hydrogen sulfide in living systems.

Hydrogen sulfide is a redox active sulfur species that is endogenously generated in mammalian systems as an antioxidant and signaling molecule to support cellular function. The fundamental and ubiquitous actions of hydrogen sulfide demand sensitive and specific methods to track this biomolecule as it is produced within living organisms with temporal and spatial regulation. In this context, the hydrogen sulfide-mediated reduction of an azide to an amine is a useful method for organic synthesis, and this reaction has successfully been exploited to yield biocompatible fluorescent probes for hydrogen sulfide detection in vitro and in cells. This chapter provides protocols and guidelines for applying azide-based fluorescence probes to detecting hydrogen sulfide in living systems, including a protocol that was used to detect endogenous hydrogen sulfide in living single cells using a confocal microscope.

¹9F magnetic resonance probes for live-cell detection of peroxynitrite using an oxidative decarbonylation reaction.

We report a newly discovered oxidative decarbonylation reaction of isatins that is selectively mediated by peroxynitrite (ONOO(-)) to provide anthranilic acid derivatives. We have harnessed this rapid and selective transformation to develop two reaction-based probes, 5-fluoroisatin and 6-fluoroisatin, for the low-background readout of ONOO(-) using (19)F magnetic resonance spectroscopy. 5-fluoroisatin was used to non-invasively detect ONOO(-) formation in living lung epithelial cells stimulated with interferon-γ (IFN-γ).

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.

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.

Lanthanide-based luminescent probes for selective time-gated detection of hydrogen peroxide in water and in living cells.

Lanthanide-based luminescent probes TPR1 and TPR2 were developed for the detection of hydrogen peroxide (H(2)O(2)) in living systems. The chemoselective reaction of these boronate-protected probes with H(2)O(2) resulted in an enhanced lanthanide sensitization and a 6-fold increase in luminescent intensity. TPR2 was utilized to measure the endogenous production of H(2)O(2) in RAW 264.7 macrophages using time-gated luminescent spectroscopy.

Stereoretentive synthesis and chemoselective amide-forming ligations of C-terminal peptide alpha-ketoacids.

C-Terminal peptide cyanosulfur ylides are readily converted to C-terminal peptide alpha-ketoacids, poised for chemoselective amide-forming reactions with hydroxylamines. These easily prepared and bench stable ylides are quickly and selectively oxidized with aqueous Oxone without the need for protection of most peptide side chains and with minimal epimerization. This approach offers the first method for preparing enantiomerically enriched, side chain unprotected alpha-ketoacids.