Structure-Activity Optimization of Phenoxy-1,2-Dioxetane Precursors as Probes for Singlet Oxygen Yields Unprecedented Detection Sensitivity

Chemiluminescence imaging has emerged as a powerful alternative to fluorescence-based methods, offering significant advantages such as reduced background noise, elimination of autofluorescence, and prevention of photobleaching. These benefits are particularly critical for singlet oxygen detection, where excitation light in fluorescence techniques can inadvertently generate singlet oxygen, compromising measurement accuracy. Despite this potential, the development of highly sensitive chemiluminescent probes for singlet oxygen detection under physiological conditions remains an urgent challenge. Here, we present a comprehensive structure-activity optimization of phenoxy-1,2-dioxetane precursors as probes for singlet oxygen detection in biologically relevant environments. By systematically evaluating key parameters—steric hindrance at the oxidation site, the chemiexcitation rate of the luminophore, and total light emission, we significantly increased the detection sensitivity of the singlet oxygen probe. Notably, a cyclobutyl-enolether probe (SOCL-CB) and a dimethyl-enolether probe (SOCL-DM) demonstrated 57-fold and 118-fold higher signal-to-noise (S/N) ratios, respectively, compared to the previously reported chemiluminescent adamantyl-enolether probe (SOCL-AD). The superior detection sensitivity of probe SOCL-DM was validated in an enzymatic model where singlet oxygen production was mediated by horseradish peroxidase. Remarkably, probe SOCL-DM detected singlet oxygen concentrations as low as 127 nM in this system, outperforming the previously reported probe SOCL-AD. These results establish SOCL-DM as the most sensitive chemiluminescent probe for singlet oxygen detection under physiological conditions reported to date. This study underscores the potential of chemiluminescent probes like SOCL-DM to facilitate real-time monitoring of singlet oxygen, providing invaluable tools for studying oxidative stress, elucidating cellular processes, and advancing diagnostic applications.