A red-emitting naphthofluorescein-based fluorescent probe for selective detection of hydrogen peroxide in living cells

Abstract

We report the synthesis, properties, and cellular application of Naphtho-Peroxyfluor-1 (NPF1), a new fluorescent indicator for hydrogen peroxide based on a red-emitting naphthofluorescein platform. Owing to its boronate cages, NPF1 features high selectivity for hydrogen peroxide over a panel of biologically relevant reactive oxygen species (ROS), including superoxide and nitric oxide, as well as excitation and emission profiles in the far-red region of the visible spectrum (>600nm). Flow cytometry experiments in RAW264.7 macrophages establish that NPF1 can report changes in peroxide levels in living cells.

Figure 1

Fluorescence response of 5 µM NPF1 to 100 µM H₂O₂. The dashed spectrum was acquired before H₂O₂ addition (dotted line) and the solid line spectra shown were acquired after 10, 20, 30, 40, 50 and 60 min incubation with H₂O₂. Spectra were acquired in 20 mM HEPES, pH 7.5, at 37 °C (λ_exc = 598 nm).

Figure 2

Fluorescence responses of 5 µM NPF1 to 100 µM reactive oxygen species (ROS). Hydrogen peroxide (H₂O₂), tert-butyl hydroperoxide (TBHP), and hypochlorite (OCl⁻) were delivered from 30%, 70%, and 5% aqueous solutions, respectively. Hydroxyl radical (^•OH) and tert-butoxy radical (^•O^tBu) were generated by reactions of 1 mM Fe²⁺ with 100 µM H₂O₂ or 100 µM TBHP, respectively. Superoxide (O₂⁻) was generated enzymatically using a xanthine/xanthine oxidase system. NO⁺ was delivered using S-nitrosocysteine (SNOC). NO was delivered using NOC-5. Spectra were acquired in 20 mM HEPES, pH 7.5, and all data were obtained after incubation with the appropriate ROS at 37 °C. Bars represent relative emission responses (λ_exc = 598 nm, λ_em = 660 nm at 0 (white), 15 (light gray), 30 (gray), 45 (dark gray), and 60 min (black) after addition of the appropriate ROS.

Figure 3

Time-course kinetics measurement of the fluorescence response of NPF1 to H₂O₂. Data were collected under pseudo-first-order conditions (1 µM NPF1, 1 mM H₂O₂). Spectra were acquired in 20 mM HEPES, pH 7.5, at 25 °C (λ_exc = 598 nm, λ_em = 660 nm), and data are plotted as relative emission intensities over initial background.

Figure 4

Flow cytometry analysis of NPF1-loaded live RAW 264.7 macrophages in response to increases in H₂O₂ levels. Two aliquots of cells were incubated with 20 µM NPF1 for 1 h. 100 µM H₂O₂ was subsequently added to one of the aliquots and the cells were incubated for an additional 1 h. Cells were then analyzed by flow cytometry. (a) Represetative flow cytometry trace from one experiment described above. Data are shown for NPF1-loaded control cells in the absence of H₂O₂ (gray) and cells treated with H₂O₂ (red). (b) Mean relative fluorescence for populations shown in panel (a) from three replicate experiments. Error bars represent the standard deviation from the mean for the three experiments. The data represent at least 10,000 cells for each analysis.

Scheme 1

Synthesis and activation of Naphtho-Peroxyfluor-1 (NPF1). Reagents and conditions: (i) N-phenyl-bis(trifluoromethanesulfonimide), DIPEA, DMF, 25 °C, 24 h. (ii) Pd(dppf)Cl₂•CH₂Cl₂, dppf, bis(pinacolato)diboron, KOAc, 1,4-dioxane, 100 °C, 24 h.