Modulation of Emission and Singlet Oxygen Photosensitization in Live Cells Utilizing Bioorthogonal Phosphorogenic Probes and Protein Tag Technology

The self-labeling protein SNAP-tag is commonly used to study the expression and functions of proteins of interest (POI) in live cells. It undergoes a specific reaction with synthetic probes containing a benzylguanine (BG) moiety. Despite the fact that many substrates for SNAP-tag have been developed, photofunctional substrates that serve as both fluorogenic probes and singlet oxygen (¹O₂) photosensitizers have not been reported. Herein, we report the design of a new class of phosphorogenic iridium(III) nitrone complexes for these purposes. In this strategy, a strained alkyne-modified substrate BCN-BG was used to label SNAP-tag, which was then reacted with the iridium(III) nitrone complexes. These complexes were weakly emissive with negligible ¹O₂ generation due to efficient quenching of the nitrone moiety. However, they displayed significant emission enhancement (I/I_o) upon reactions with the strained alkyne BCN-OH (I/I_o = ca. 7.2 – 47.1) and BCN-modified BSA (I/I_o = ca. 82.9 – 327.1). These reactions also resulted in excellent photoinduced ¹O₂ generation (generation quantum yields up to 0.91). Confocal images revealed that CHO-K1 cells expressing organelle-specific SNAP-tag exhibited intense and long-lived emission only upon treatment with both BCN-BG and the complexes. MTT assays indicated that BCN-BG and SNAP-tag significantly increased the photocytotoxicity of the complexes, resulting in a photocytotoxicity index up to 115.9. We believe that this two-step labeling approach will lead to the development of new theranostic applications.