Conspectus 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 H2O2, 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 H2O2 and other ROS have essential functions in healthy, physiological signaling pathways. The signal–stress dichotomy of H2O2 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 H2O2 chemistry in biological specimens, but two key challenges to studying H2O2 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 H2O2 studies from our laboratory using the H2O2-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 H2O2 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 H2O2 in a wide variety of living systems, including immune, cancer, stem, and neural cell models.
Living organisms produce hydrogen peroxide (H 2 O 2 ) to kill invading pathogens and for cellular signaling, but aberrant generation of this reactive oxygen species is a hallmark of oxidative stress and inflammation in aging, injury, and disease. The effects of H 2 O 2 on the overall health of living animals remain elusive, in part owing to a dearth of methods for studying this transient small molecule in vivo. Here we report the design, synthesis, and in vivo applications of Peroxy Caged Luciferin-1 (PCL-1), a chemoselective bioluminescent probe for the real-time detection of H 2 O 2 within living animals. PCL-1 is a boronic acid-caged firefly luciferin molecule that selectively reacts with H 2 O 2 to release firefly luciferin, which triggers a bioluminescent response in the presence of firefly luciferase. The high sensitivity and selectivity of PCL-1 for H 2 O 2 , combined with the favorable properties of bioluminescence for in vivo imaging, afford a unique technology for real-time detection of basal levels of H 2 O 2 generated in healthy, living mice. Moreover, we demonstrate the efficacy of PCL-1 for monitoring physiological fluctuations in H 2 O 2 levels by directly imaging elevations in H 2 O 2 within testosterone-stimulated tumor xenografts in vivo. The ability to chemoselectively monitor H 2 O 2 fluxes in real time in living animals offers opportunities to dissect H 2 O 2 ’s disparate contributions to health, aging, and disease.
In vivo molecular imaging holds promise for understanding the underlying mechanisms of health, injury, aging, and disease, as it can detect distinct biochemical processes such as enzymatic activity, reactive small-molecule fluxes, or post-translational modifications. Current imaging techniques often detect only a single biochemical process, but, within whole organisms, multiple types of biochemical events contribute to physiological and pathological phenotypes. In this report, we present a general strategy for dual-analyte detection in living animals that employs in situ formation of firefly luciferin from two complementary caged precursors that can be unmasked by different biochemical processes. To establish this approach, we have developed Peroxy Caged Luciferin-2 (PCL-2), a H2O2-responsive boronic acid probe that releases 6-hydroxy-2-cyanobenzothiazole (HCBT) upon reacting with this reactive oxygen species (ROS), as well as a peptide-based probe, Ile-Glu-Thr-Asp-D-Cys (IETDC) which releases D-cysteine in the presence of active caspase 8. Once released, HCBT and D-cysteine form firefly luciferin in situ, giving rise to a bioluminescent signal if and only if both chemical triggers proceed. This system thus constitutes an AND-type molecular logic gate that reports on the simultaneous presence of H2O2 and caspase 8 activity. Using these probes, chemoselective imaging of either H2O2 or caspase 8 activity was performed in vitro and in vivo. Moreover, concomitant use of PCL-2 and IETDC in vivo establishes a concurrent increase in both H2O2 and caspase 8 activity during acute inflammation in living mice. Taken together, this method offers a potentially powerful new chemical tool for studying simultaneous oxidative stress and inflammation processes in living animals during injury, aging, and disease, as well as a versatile approach for concurrent monitoring of multiple analytes using luciferin-based bioluminescence imaging technologies.
Copper is a required metal nutrient for life, but global or local alterations in its homeostasis are linked to diseases spanning genetic and metabolic disorders to cancer and neurodegeneration. Technologies that enable longitudinal in vivo monitoring of dynamic copper pools can help meet the need to study the complex interplay between copper status, health, and disease in the same living organism over time. Here, we present the synthesis, characterization, and in vivo imaging applications of Copper-Caged Luciferin-1 (CCL-1), a bioluminescent reporter for tissue-specific copper visualization in living animals. CCL-1 uses a selective copper(I)-dependent oxidative cleavage reaction to release d-luciferin for subsequent bioluminescent reaction with firefly luciferase. The probe can detect physiological changes in labile Cu+ levels in live cells and mice under situations of copper deficiency or overload. Application of CCL-1 to mice with liver-specific luciferase expression in a diet-induced model of nonalcoholic fatty liver disease reveals onset of hepatic copper deficiency and altered expression levels of central copper trafficking proteins that accompany symptoms of glucose intolerance and weight gain. The data connect copper dysregulation to metabolic liver disease and provide a starting point for expanding the toolbox of reactivity-based chemical reporters for cell- and tissue-specific in vivo imaging.
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