A Chemiluminescent Probe for HNO Quantification and Real-Time Monitoring in Living Cells

Abstract

Azanone (HNO) is a reactive nitrogen species with pronounced biological activity and high therapeutic potential for cardiovascular dysfunction. A critical barrier to understanding the biology of HNO and furthering clinical development is the quantification and real-time monitoring of its delivery in living systems. Herein, we describe the design and synthesis of the first chemiluminescent probe for HNO, HNOCL-1, which can detect HNO generated from concentrations of Angeli's salt as low as 138 nm with high selectivity based on the reaction with a phosphine group to form a self-cleavable azaylide intermediate. We have capitalized on this high sensitivity to develop a generalizable kinetics-based approach, which provides real-time quantitative measurements of HNO concentration at the picomolar level. HNOCL-1 can monitor dynamics of HNO delivery in living cells and tissues, demonstrating the versatility of this method for tracking HNO in living systems.

Keywords: bioanalysis; chemiluminescence; nitroxyl; phosphine.

Figure 1.

Chemiluminescence response of HNOCL-1 and Angeli’s salt, Na₂N₂O₃ (AS). (A) Chemiluminescence emission spectra at 525 nm of 20 μM HNOCL-1 and 0 (blue trace), 50, 100, 150, and 200 μM (red trace) AS. (B) Time course of chemiluminescence emission of 0 (blue trace), 5, 25, 50, and 100 μM (red trace) AS. (C) Time course of chemiluminescence emission of 10 μM HNOCL-1 and 0 (blue trace) or 200 μM (red trace) AS. (D) Fluorescence emission spectra of 10 μM XF2 after reacting with 0 (blue trace) or 200 μM (red trace) AS for 25 minutes. (E)–(F) Concentration of HNO generated from AS (E) measured from the raw chemiluminescence emission of 20 μM HNOCL-1 or (F) computationally simulated. All experiments were performed in 20 mM HEPES or PBS (pH 7.4), containing ≤1% DMSO. Error bars are ± S.D. from n = 6 replicates.

Figure 2.

Chemiluminescent responses of 20 μM HNOCL-1 and 200 μM RSON species. Legend: 1. AS, 2. GSH (2 mM), 3. GSNO, 4. H₂O₂, 5. KO₂, 6. Cys (1 mM), 7. Na₂S, 8. Na₂S₂O₃, 9. NaNO₂, 10. HO^•, 11. ONOO^–, 12. ^tBuOOH, 13. OCl^–, 14. DEA NONOate, 15. Blank. (B) Time-course of the chemiluminescent emission of 20 μM HNOCL-1 alone (blue trace), with 1 mM DEA NONOate (DN) in PBS (black trace), and with 1 mM DN in HEPES (red trace). (C) Time course of chemiluminescent emission and (D) integrated emission intensity of 20 μM HNOCL-1 alone (blue trace), 200 μM Na₂S alone or with 0.2, 0.5, and 1 mM (red trace) DN, and 200 μM Na₂S, 1 mM DN, and 2 mM N-acetyl cysteine (NAC). (E) Concentration of HNO produced from 20 μM HNOCL-1 alone (blue trace), and 200 μM Na₂S and 0.2, 0.5, and 1 mM (red trace) DN as determined from Eq. (1) and the data shown in (C). All experiments were performed in 20 mM HEPES or PBS (pH 7.4), containing ≤1% DMSO. Error bars are ± S.D. from n = 3–6 replicates.

Figure 3.

Chemiluminescent measurement of HNO in living cells. (A) Time course of chemiluminescent emission and (B) integrated emission intensity of A549 cells incubated with 20 μM HNOCL-1 for 30 minutes, washed and then treated with AS. Error bars are ±S.D. from n = 9 wells across 3 biological replicates. (C) Time course of chemiluminescence emission of A549 cells incubated with 20 μM HNOCL-1 for 30 minutes, washed and then incubated without (blue trace) or with 1 mM DEA NONOate (DN) and 200 μM Na₂S (red trace). (D) Integrated emission intensity of A549 cells incubated with 20 μM HNOCL-1 for 30 minutes, washed and then treated as indicated with H₂S as 200 μM Na₂S, NO as 1 mM DN, and NAC at 2 mM. Error bars are ±S.E. from n = 6–11 wells and 2–4 biological replicates. Statistical significance was assessed using a two-tailed student’s t-test. **p<0.01, *p<0.05. All cellular experiments were performed at 37 °C. (E)–(F) Images of BALB/c nude mice 2 minutes after IP injection with 40 μM HNOCL-1 and (E) vehicle control or (F) 1 mM Angeli’s salt in 20 mM PBS (pH 7.4) containing 5% DMSO.

Scheme 1.

Design and synthesis of the chemiluminescent HNO probe HNOCL-1.