Phenoxy-1,2-dioxetane-based activatable chemiluminescent probes: tuning of photophysical properties for tracing enzymatic activities in living cells

The use of chemiluminophores for tracing enzymatic activities in live-cell imaging has gained significant attention, making them valuable tools for diagnostic applications. Among various chemiluminophores, the phenoxy-1,2-dioxetane scaffold exhibits significant structural versatility and its activation is governed by the chemically initiated electron exchange luminescence (CIEEL) mechanism. This mechanism can be initiated by enzymatic activity, changes in pH, or other chemical stimuli. The photophysical properties of phenoxy-1,2-dioxetanes can be fine-tuned through the incorporation of different substituents on the phenolic ring and by anchoring them with specific triggers. This review discusses the variations in physicochemical properties, including emission maxima, quantum yield, aqueous solubility, and pKa, as influenced by structural modifications, thereby establishing a comprehensive structure–activity relationship. Furthermore, it categorises the probes based on different enzyme classes, such as hydrolase-sensitive probes, oxidoreductase-responsive probes, and transferase-activatable phenoxy-1,2-dioxetanes, offering a promising platform technology for the early diagnosis of diseases and disorders. The summary section highlights key opportunities and limitations associated with applying phenoxy-1,2-dioxetanes in achieving precise and effective enzyme assays.