A Selective, Cell-Permeable Optical Probe for Hydrogen Peroxide in Living Cells

Hydrogen peroxide is a major reactive oxygen species (ROS) in living organisms, and its homeostasis can have diverse physiological and pathological consequences.¹ H₂O₂ is a source of oxidative stress,² and oxidative damage resulting from cellular imbalance of H₂O₂ and other ROS oxidants is connected to aging and severe human diseases such as cancer,³ cardiovascular disorders,⁴ and Alzheimer's and related neurodegenerative diseases.⁵ On the other hand, emerging evidence supports a physiological role for H₂O₂ as a second messenger in cellular signal transduction.^6-8 For example, peroxide bursts trigger mitogen-activated protein (MAP) kinase⁹ and nuclear factor κB (NF-κB)¹⁰ pathways that affect cell proliferation and cell death.

Despite the importance of H₂O₂ to human health and disease, the molecular mechanisms of its production, accumulation, trafficking, and function are insufficiently understood even in the simplest eukaryotic organisms.² We are interested in developing new chemical tools to study the physiological and pathological roles of H₂O₂ and related ROS in living systems. In this regard, fluorescent probes are well suited to meet the need for reagents to interrogate the cellular chemistry of H₂O₂ at the molecular level. One major challenge to achieving this goal is creating water-soluble systems that report H₂O₂ selectively over competing cellular ROS like superoxide (O₂⁻), nitric oxide (NO), and lipid alkylperoxides. Synthetic small molecules offer one approach to such probes, and several types of reagents have been examined for H₂O₂ detection. Included are dihydro analogs of fluorescent dyes (e.g., 2′,7′-dichlorodihydrofluorescein (DCFH), Amplex Red, dihydrorhodamine 123),^11-13 phosphine-derivatized fluorophores,^14,15 lanthanide coordination complexes,¹⁶ and chromophores with ROS-cleavable protecting groups.^17-19 However, limitations of currently available H₂O₂-responsive probes include interfering background fluorescence from other ROS, the need for an external activating enzyme, lack of water solubility or compatibility, and/or excitation profiles in the ultraviolet region, which can damage living samples and cause interfering autofluorescence from native cellular species. The most commonly used fluorophore for cellular ROS detection, DCFH, is also easily autoxidized and exhibits increased background fluorescence upon continued exposure to light.¹⁷ In this report, we present the synthesis and properties of Peroxyfluor-1 (PF1, 2), a new water-soluble, turn-on optical probe for H₂O₂ that exhibits high selectivity and dynamic range for this small molecule over other ROS, and establish its utility for imaging changes in [H₂O₂] within living mammalian cells with visible wavelength excitation and emission energies.

Our strategy for the optical detection of H₂O₂ relies on the selective H₂O₂-mediated transformation of arylboronates to phenols.²⁰ In particular, we reasoned that installation of boronic ester groups at the 3′ and 6′ positions of a xanthenone scaffold would force this platform to adopt a closed, colorless, and non-fluorescent lactone form. Upon treatment with H₂O₂, hydrolytic deprotection of the boronates would subsequently generate the open, colored, and fluorescent fluorescein product. Scheme 1 outlines the preparation of PF1 based on this design. Acid-catalyzed condensation of 3-iodophenol and phthalic anhydride affords 3′,6′-diiodofluoran 1 in 25% yield.²¹ Palladium-catalyzed transmetallation of fluoran 1 under Masuda conditions with bis(pinacolato)diboron proceeds smoothly to generate PF1 in 50% yield after workup and purification by column chromatography.

Scheme 1.

Synthesis of Peroxyfluor-1 (PF1).

PF1 was evaluated under simulated physiological conditions (20 mM HEPES buffer, pH 7). As expected, the parent compound is non-fluorescent and displays no absorption features in the visible region. The addition of H₂O₂ triggers a prompt fluorescence increase (Figure 1A) with concomitant growth of a visible wavelength absorption band characteristic of fluorescein. Absorption and emission spectra, along with electrospray ionization mass spectrometry, confirm that fluorescein is the product generated from the reaction between PF1 and H₂O₂. The dynamic range of this probe is large owing to its binary absorption/emission response. The fluorescence response of PF1 is also highly H₂O₂ selective. Figure 1B compares the relative reactivities of PF1 toward various ROS. PF1 exhibits a greater than 500-fold higher response for H₂O₂ over similar ROS such as tert-butyl hydroperoxide (TBHP), O₂⁻, NO, or ⁻OCl, which represents a 10 to 100-fold increase in H₂O₂ selectivity compared to previously reported probes.^11,18,19 The xanthenone probe is also more responsive to H₂O₂ compared to highly reactive oxygen radicals such as •O^tBu (> 15-fold higher for H₂O₂) and •OH (> 3-fold higher for H₂O₂). We suggest that the observed selectivity for H₂O₂ over more oxidizing ROS is based on the detection mechanism of PF1, which relies on deprotection rather than oxidation to provide an optical response.

Figure 1.

(A) Fluorescence response of 5 μM PF1 to 100 μM H₂O₂. The dotted and solid line spectra were recorded before and after H₂O₂ addition, respectively. Spectra were acquired in 20 mM HEPES, pH 7 (λ_exc = 450 nm). (B) Fluorescence responses of 5 μM PF1 to various ROS (10 mM O₂⁻, 100 μM for all other ROS). •OH and •O^tBu were generated by reaction of Fe²⁺ with H₂O₂ or TBHP, respectively. NO was delivered using S-nitrosocysteine (SNOC). Spectra were acquired in 20 mM HEPES, pH 7, and all data were obtained after incubation with the appropriate ROS at 25 °C for 1 h. Collected emission was integrated between 460 and 700 nm (λ_exc = 450 nm).

We next assessed the ability of PF1 to operate within living cells. HEK cells were incubated with 5 μM PF1 for 5 min at 25 °C and show negligible intracellular background fluorescence (Figure 2A). Prompt increases in cytosolic fluorescence are observed upon addition of physiologically relevant concentrations of exogenous H₂O₂ (10-100 μM, Figure 2B) as determined from scanning confocal microscopy on live samples. Control experiments performed without dye or H₂O₂ give negligible fluorescence responses, and transmittance measurements after PF1 incubation and H₂O₂ addition (Figure 2C) confirm that the cells are viable throughout the imaging experiments. These data establish that PF1 is membrane-permeable and can respond to μM changes in H₂O₂ concentrations within living cells. In addition, subsequent experiments show that fluorescence responses down to 100 nM H₂O₂ are readily detectible in vitro.

Figure 2.

Confocal fluorescence and phase contrast images of live HEK cells. (A) Fluorescence image of HEK cells incubated with 5 μM PF1 for 5 min at 25 °C. (B) Fluorescence image of PF1-stained HEK cells treated with 100 μM H₂O₂ for 5 min at 25 °C. (C) Brightfield image of live HEK cells after H₂O₂ addition to confirm viability. Scale bar = 30 μm.

To close, we have presented the synthesis, properties, and biological applications of PF1, a new type of probe for optical imaging of intracellular H₂O₂. This fluorescein-based reagent features excellent selectivity for H₂O₂ over competing cellular ROS, large dynamic response range owing to its dual colorimetric/fluorometric detection mechanism, and long-wavelength visible excitation and emission profiles to minimize cell and tissue damage while avoiding interfering autofluorescence from native cellular species. Furthermore, we have demonstrated the value of this probe by measuring changes in intracellular [H₂O₂] within living mammalian cells. Current efforts are directed toward applying PF1 and related tools for studying the oxidation biology of living systems.

We present the synthesis, properties, and biological applications of Peroxyfluor-1 (PF1), a new type of optical probe for intracellular imaging of hydrogen peroxide in living biological samples. PF1 utilizes a boronate deprotection mechanism to provide unprecedented selectivity and optical dynamic range for detecting H₂O₂ in aqueous solution over similar reactive oxygen species including superoxide, nitric oxide, tert-butyl hydroperoxide, and hydroxyl radical. We further demonstrate the value of this reagent for biological applications by imaging changes in [H₂O₂] in living mammalian cells.