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

Abstract

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.

Figure 1

Unregulated production of ROS such as H₂O₂ can result in oxidative damage, but these molecules also play central roles in protein folding, signaling, defense response, and respiration and metabolism.

Figure 2

Design of a bioorthogonal reactivity approach for selective H₂O₂ detection via boronate oxidation.

Figure 3

Boronate-based probes for H₂O₂ detection and imaging.

Figure 4

(a) Fluorescence response of Peroxyfluor 1 (PF1) to H₂O₂. The dashed and solid spectra were recorded before and after H₂O₂ addition, respectively. (b) Fluorescence responses of PF1 to various ROS. Bars represent relative responses after 5, 15, 30, 45, and 60 min after addition of the given ROS.

Figure 5

Images of Peroxyresorufin 1 (PR1), Peroxyfluor 1 (PF1), and Peroxyxanthone 1 (PX1) detecting H₂O₂ fluxes in living cells.

Figure 6

H₂O₂ and growth factor signaling in living neurons. EGF stimulation produces an increase in the fluorescence response of Peroxy Green 1 (PG1). This response is attenuated by apocynin, a Nox inhibitor; wortmannin, an inhibitor of PI3K; PD153035, an inhibitor of the receptor tyrosine kinase domain of the EGF receptor; and NSC23766, a Rac1 inhibitor.

Figure 7

Confocal fluorescence images of H₂O₂-producing phagosomes, hROS-producing phagosomes, and dual H₂O₂ and hROS-producing phagosomes in live RAW264.7 macrophages as distinguished by simultaneous imaging with Peroxy Orange 1 (PO1) and Aminophenyl Fluorescein (APF).

Figure 8

Ratiometric confocal fluorescence images of H₂O₂ in stimulated live RAW 264.7 macrophages as visualized with Peroxy Lucifer 1 (PL1).

Figure 9

Images of SNAP Peroxy Green 2 (SPG2) localized to the plasma membrane, mitochondria, endoplasmic reticulum, and nucleus.

Figure 10

Aquaporins facilitate H₂O₂ trafficking in growth factor signaling, as shown by the aquaglyceroporin family isoform Aquaporin 3 (AQP3).

Figure 11

FGF stimulation induces a Nox2-dependent increase in intracellular H₂O₂ levels as imaged by Peroxyfluor 6 (PF6).

Figure 12

Peroxy Caged Luciferin 1 (PCL-1) detects H₂O₂ in vivo using bioluminescence.