Rational Design of a Dual-Reactivity-Based Fluorescent Probe for Visualizing Intracellular HSNO

Abstract

Thionitrous acid (HSNO), the smallest S-nitrosothiol, is emerging as a potential key intermediate in cellular redox regulation linking two signaling molecules H₂ S and NO. However, the chemical biology of HSNO remains poorly understood. A major hurdle is the lack of methods for selective detection of HSNO in biological systems. Herein, we report the rational design, synthesis, and evaluation of the first fluorescent probe TAP-1 for HSNO detection. TAP-1 showed high selectivity and sensitivity to HSNO in aqueous media and cells, providing a useful tool for understanding the functions of HSNO in biology.

Keywords: S-nitrosothiols; biosensors; fluorescent probes; hydrogen sulfide; imaging agents.

Figure 1.

(a) Turn-on fluorescence response of 10 μM TAP-1 to 6-fold diluted HSNO solution at different reaction time (0, 1, 2, 4, 6, 8, 10 min for curves 1–7, respectively); (b) Fluorescence intensity at 521 nm of 10 μM TAP-1 in the presence of various reactive species: 1) blank; 2) 10 mM GSH; 3) 1 mM Cys; 4) 1 mM Hcy; 5) 200 μM Na₂S; 6) 100 μM Na₂SO₃; 7) 100 μM MCPD; 8) 100 μM Na₂S₂; 9) 100 μM Na₂S₃; 10) 1 mM GSNO; 11) 200 μM pyrrolidine-NONOate; 12) 100 μM Angeli’s salt; 13) 100 μM ONOO⁻; 14) 100 μM t-BuONO; 15) 1 mM NaNO₂; 16) 1 mM KNO₃; 17) 50 μM Na₂S + 50 μM pyrrolidine-NONOate; 18) HSNO solution (50 μM).

Figure 2.

Fluorescence images of TAP-1 in HeLa cells. (a-f) Cells were incubated with 10 μM TAP-1 for 30 min, washed and subjected to different treatments for 25 min. a) Controls (only FBS-free media); b) 150 μM Na₂S; c) 1 mM GSNO; d) 100 μM HSNO solution; e) the mixture solution of 150 μM pyrrolidine-NONOate and 150 μM Na₂S; f) 150 μM pyrrolidine-NONOate. (g) Cells were pre-treated with 1 mM PAG for 1.5 h, washed and subjected to same treatment with f). (h) Mean fluorescence intensities of images a-g. Scale bar represents 50 μm for all images.

Figure 3.

(a) Fluorescence images of TAP-1 for endogenous HSNO in HEK293T cells. WT and CARS2 KO HEK293T cells were incubated with 10 μM of TAP-1 for 30 min, washed, and treated with 10, 100 and 300 μM of NOC7. (b) Mean fluorescence intensities in panel a. Scale bar represents 50 μm for all images. Values are means ± s.d. (n = 3). ***p < 0.001 (vs. WT HEK293T), two-way ANOVA followed by Tukey-Kramer test.

Scheme 1.

Design of dual-reactivity based probe TAP-1.

Scheme 2.

Synthesis of TAP-1. Reagents and conditions: (i) SOCl₂, Et₃N, N-Boc-1,2-phenylenediamine, rt, 1 h; (ii) LiOH, THF, H₂O, rt, 4h; (iii) BH₃/THF, reflux, 4 h; (iv) chloranil, CH₂Cl₂, rt, 2 h; (v) 2-(2-pyridinyldithio)benzoic acid, EDC, DMAP, CH₂Cl₂, rt, 3 h; (vi) HCl gas, CH₂Cl₂, rt, 2 h; (vii) PPh₃, THF/H₂O, rt, 1 h.