A chemiluminescent probe for cellular peroxynitrite using a self-immolative oxidative decarbonylation reaction

Abstract

Peroxynitrite (ONOO^-) is a highly reactive oxygen species which has been recognized as an endogenous mediator of physiological activities like the immune response as well as a damaging agent of oxidative stress under pathological conditions. While its biological importance is becoming clearer, many of the details of its production and mechanism of action remain elusive due to the lack of available selective and sensitive detection methods. Herein, we report the development, characterization, and biological applications of a reaction-based chemiluminescent probe for ONOO^- detection, termed as PNCL. PNCL reacts with ONOO^-via an isatin moiety through an oxidative decarbonylation reaction to initiate light emission that can be observed instantly with high selectivity against other reactive sulphur, oxygen, and nitrogen species. Detailed studies were performed to study the reaction between isatin and ONOO^-, which confirm selectivity for ONOO^- over NO₂˙. PNCL has been applied for ONOO^- detection in aqueous solution and live cells. Moreover, PNCL can be employed to detect cellular ONOO^- generated in macrophages stimulated to mount an immune response with lipopolysaccharide (LPS). The sensitivity granted by chemiluminescent detection together with the specificity of the oxidative decarbonylation reaction provides a useful tool to explore ONOO^- chemistry and biology.

Scheme 1. Spiroadamantane 1,2-dioxetane-based probe for chemiluminescent ONOO^– detection.

Scheme 2. Synthesis of PNCL.

Fig. 1. Response of PNCL to ONOO^–. (A) Chemiluminescent emission spectra of 20 μM PNCL and 0 (blue trace), 5, 10, 20, 40, 80, 100, 200 μM (red trace) ONOO^–. (B) Time scans of the chemiluminescent emission at 525 nm of 20 μM PNCL and 0 (blue trace), 5, 10, 20, 40, 80, 100, 200 μM (red trace) ONOO^–. (C) Peak emission intensity at 525 nm of 20 μM PNCL after adding 0–200 μM ONOO^–. (D) Time scan of the chemiluminescent emission at 525 nm of 20 μM PNCL and 200 μM SIN-1. All experiments were performed in 20 mM HEPES (pH 7.4), containing <1% DMSO.

Fig. 2. Selectivity of PNCLversus cations and reactive sulphur, oxygen, and nitrogen species. Chemiluminescent emission at 525 nm of 20 μM PNCL and 200 μM biologically relevant analytes in 20 mM HEPES (pH 7.4). Bars represent chemiluminescent emission at 525 nm at 4, 8, 12, 16, 20 min after addition of reactive species. Legend: (1) ONOO^–, (2) Cys, (3) DEA NONOate, (4) GSH (5 mM), (5) GSNO, (6) H₂O₂, (7) Angeli's salt, (8) KO₂, (9) Na₂S, (10) Na₂S₂O₃, (11) Na₂SO₄, (12) NaNO₂, (13) HO˙, (14) OCl^–, (15) ^tBuOOH, (16) ¹O₂, (17) Na⁺, (18) Mg²⁺, (19) K⁺, (10) Ca²⁺, (21) Zn²⁺, (21) Fe²⁺, (23) blank. Error bars are ± S.D. All experiments were performed in 20 mM HEPES or 20 mM PBS (pH 7.4), containing <1% DMSO.

Fig. 3. Inhibition of response by NaHCO₃. (A) Time scans and (B) integrated intensity of the chemiluminescent emission of 20 μM PNCL, 200 μM ONOO^– and 0 (blue trace), 1 mM, 2 mM, 3 mM, 4 mM, and 5 mM (red trace) NaHCO₃. (C) Time scans and (D) integrated intensity of the chemiluminescent emission of 20 μM PNCL, 200 μM Angeli's salt and 0 (blue trace), 1 mM, 2 mM, 3 mM, 4 mM, and 5 mM (red trace) NaHCO₃. All experiments were performed in 20 mM HEPES (pH 7.4), containing <1% DMSO.

Fig. 4. Inhibition of response by glutathione. (A) Time scans and (B) integrated intensity of the chemiluminescent emission of 20 μM PNCL, 200 μM ONOO^– and 0 (blue trace), 50 μM, 100 μM, 200 μM, and 1 mM (red trace) glutathione. (C) Time scans and (D) integrated intensity of the chemiluminescent emission of 20 μM PNCL, 200 μM Angeli's salt and 0 (blue trace), 50 μM, 100 μM, 200 μM, and 1 mM (red trace) glutathione. All experiments were performed in 20 mM HEPES (pH 7.4), containing <1% DMSO.

Fig. 5. HNO scavenging by NaHCO₃ and GSH. (A) Design of XF1, a fluorescent probe for HNO. (B) Peak emission intensity of 10 μM XF1, 200 μM Angeli's salt and 0–5 mM NaHCO₃. (C) Peak emission intensity of 10 μM XF1, 200 μM Angeli's salt and 0–1 mM glutathione. All experiments were performed in 20 mM HEPES (pH 7.4), containing <1% DMSO with λ_ex = 488 nm.

Fig. 6. ONOO^– detection in RAW 264.7 macrophages. (A) Time scans and (B) integrated intensity of chemiluminescence emission of RAW 264.7 macrophages incubated with 20 μM PNCL for 30 minutes, washed, and then treated with 0 (blue trace), 200 μM, 400 μM, 800 μM, 1 mM, and 2 mM (red trace) SIN-1. (C) Integrated chemiluminescent emission intensity of RAW 264.7 macrophages stimulated with (Cont) Vehicle control, (LPS) 1000 ng mL^–1 LPS, or (LPS, MnTMPyP) 1000 ng mL^–1 LPS and 25 μM Mn(iii)TMPyP and then treated with 20 μM PNCL. (D) Brightfield images of stimulated RAW 264.7 macrophages. Error bars are S.D. from n = 3–7 wells and 3 biological replicates (n = 10 wells from 6 biological replicates for the control experiment). Statistical significance was assessed using a two-tailed student's t-test. ***p < 0.001, **p < 0.01.