Benzylic Trifluoromethyl Accelerates 1,6-Elimination Toward Rapid Probe Activation

Abstract

Activity-based detection of hydrogen sulfide in live cells can expand our understanding of its reactivity and complex physiological effects. We have discovered a highly efficient method for fluorescent probe activation, which is driven by H₂S-triggered 1,6-elimination of an α-CF₃-benzyl to release resorufin. In detecting intracellular H₂S, 4-azido-(α-CF₃)-benzyl resorufin offers significantly faster signal generation and improved sensitivity compared to 4-azidobenzyl resorufin. Computed free energy profiles for the 1,6-elimination process support the hypothesis that a benzylic CF₃ group can reduce the activation energy barrier toward probe activation. This novel probe design allows for near-real-time detection of H₂S in HeLa cells under stimulation conditions.

Figure 1.. The concept of α-trifluoromethylation in 4-azidobenzyl-caged probe design.

Traditional 4-azidobenzyl group undergoes a slow 1,6-elimination process (top), whereas the 4-azido-(α-CF₃)-benzyl delivers a significantly faster 1,6-elimination, allowing for rapid probe activation (bottom).

Figure 2.

The chemical syntheses of the benzyl-caged resorufin 7 and α-CF₃-benzyl-caged resorufin 8.

Figure 3.. Probe performance in vitro.

Activation kinetics (A-B), Specificity (C-D) and limit of detection (LoD) (E-F) of 7 versus 8. LoD measurements were performed in Tris buffer (100 mM, pH 7.5). Error bars represent the standard deviation; n = 3. Single-tailed Student’s t-test: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 4.. Computed free energy profiles for the 1,6-elimination of the caged resorufins 11 and 12.

All free energies were calculated under 1 atmospheric pressure and 298 K, and are reported in units of kcal/mol. Atomic representations in the molecular structures are C: grey, O: red, N: blue, F: green, and H: white.

Figure 5.. Confocal imaging of H₂S/HS⁻ using probe 8 in live HeLa cells.

Cells were incubated with probe 8 (20 μM) for 30 minutes and stained with actin dye (CellMask^™ Deep Red Actin Tracking Stain) prior to imaging. (Top row) Cells were left untreated. (Middle row) Cells were treated with Na₂S (200 μM) as the exogeneous source H₂S/HS^–. (Bottom row) Cells were treated with l-Cys (1 mM) as the substrate to induce endogenous generation of H₂S/HS^–. Cells were imaged after 1 hour following treatment with sulfur species. Resorufin channel: 571/583 nm; Actin channel: 652/669 nm. Scale bar: 20 µm.

Figure 6.. Confocal imaging of time-dependent capturing of H₂S/HS⁻ generation in stimulated HeLa cells.

Cells were incubated with either (A) probe 7 (20 μM) or (B) 8 (20 μM) and then stained with actin dye (CellMask^™ Deep Red Actin Tracking Stain) prior to imaging. Cells were treated with l-Cys (1 mM) as a substrate to CSE and CBS to induce endogenous H₂S/HS^–. Apoptosis is visible in both images at the 10 min mark and necrosis of the majority of cells by 30 min. Resorufin channel: 571/583 nm; Actin channel: 652/669 nm. Scale bar: 20 µm.