A fast and selective near-infrared fluorescent sensor for multicolor imaging of biological nitroxyl (HNO)

Abstract

The first near-infrared fluorescent turn-on sensor for the detection of nitroxyl (HNO), the one-electron reduced form of nitric oxide (NO), is reported. The new copper-based probe, CuDHX1, contains a dihydroxanthene (DHX) fluorophore and a cyclam derivative as a Cu(II) binding site. Upon reaction with HNO, CuDHX1 displays a five-fold fluorescence turn-on in cuvettes and is selective for HNO over thiols and reactive nitrogen and oxygen species. CuDHX1 can detect exogenously applied HNO in live mammalian cells and in conjunction with the zinc-specific, green-fluorescent sensor ZP1 can perform multicolor/multianalyte microscopic imaging. These studies reveal that HNO treatment elicits an increase in the concentration of intracellular mobile zinc.

Scheme 1. Possible Biosynthetic Pathways to HNO

l-NHA = N-hydroxy-l-arginine; nNOS = neuronal nitric oxide synthase; SOD = superoxide dismutase; THB = tetrahydrobiopterin.

Chart 1. Chemical Structures and Photophysical Properties of Some Previously Reported and Newly Developed (CuDHX1) Probes for HNO⁻

Scheme 2. Synthesis of 3 and DHX1

Reagents and conditions: (a) Et₃N, DMF, 110 °C, 20 min, 78%; (b) SOCl₂, pyridine, CH₂Cl₂, DMF, 0 °C, 30 min; (c) Cyclam, DIPEA, CH₃CN, reflux, 30 min, 18% (over steps b and c); (d) Benzyl bromide, DIPEA, CH₃CN, 25 °C, 2 h, 28%. Counterions are omitted for clarity. DIPEA = diisopropylethylamine.

Figure 1

Fluorescence (dotted lines) and absorbance (solid lines) spectra of DHX1 (black lines) and CuDHX1 (red lines) in aqueous buffer (50 mM PIPES, 100 mM KCl, pH = 7).

Figure 2

Fluorescence spectra of 2 μM CuDHX1 (black dashed line) in aqueous buffer (50 mM PIPES, 100 mM KCl, pH = 7) and 2 min after the addition of 100 equiv of Angeli’s salt (red solid line). The black solid line is the spectrum of ligand DHX1. λ_ex: 650 nm.

Figure 3

Normalized integrated fluorescence intensity (660–900 nm) of 2 μM CuDHX1 in aqueous buffer (50 mM PIPES, 100 mM KCl, pH = 7) and 10 min after addition of 100 equiv of the analyte or 2 min after addition of 100 equiv of HNO. λ_ex: 650 nm.

Figure 4

X-band EPR spectra of 400 μM CuDHX1 in CH₃OH. Top: CuDHX1 before (black line) and after addition of 5000 equiv of NO (red line). Middle: CuDHX1 before (black line) and after (red line) addition of 100 equiv of Angeli’s salt. Bottom: CuDHX1 after reduction by HNO (black line) and after reoxidation by air (red line). Collection parameters: temperature, 77 K; modulation amplitude, 20 G; microwave power, 0.2 mW at 9.23 GHz.

Figure 5

Fluorescence microscopy images of cells incubated with CuDHX1 in PBS before and after addition of Angeli’s salt: (A) Differential interference contrast (DIC) image; (B) blue channel showing nuclei; (C) NIR channel before addition of Angeli’s salt; and (D) NIR channel 5 min after treatment with 1.5 mM Angeli’s salt. Scale bar =25 μm.

Figure 6

Multicolor/multianalyte imaging of HNO and mobile zinc. (A) DIC image; (B) blue channel showing nuclei; (C) quantification of the fluorescence intensity in the green and NIR channels; (D) green channel before addition of Angeli’s salt; (E) green channel after addition of Angeli’s salt; (F) green channel after addition of TPEN; (G) NIR channel before addition of Angeli’s salt; (H) NIR channel after addition of Angeli’s salt; and (I) NIR channel after addition of TPEN. Scale bar =10 μm.