A 1,2-Dioxetane-Based Chemiluminescent Probe for In Vivo Copper Imaging

Copper is involved in healthy physiology and aberrant pathology with prominent roles in Wilson's and Menkes diseases. Tracking its reactivity in living systems is crucial to understanding its biological function and could be helpful in identifying targeted therapies. We developed a turn-on chemiluminescent copper probe CpCL, which was designed by linking a picolinic acid moiety to the phenolic substituent of a spiroadamantane 1,2-dioxetane. Reaction of CpCL with copper results in formation of a copper complex intermediate that releases the phenolate upon hydrolysis, followed by initiation of a chemiluminescence reaction for light emission. The probe displayed excellent selectivity for Cu2+ compared to other competitive metal ions, with a sensitive limit of detection of 360 nM. The mechanism was studied using initial rate kinetics to provide an estimated pseudo-first-order rate constant of 5.18 ± 0.38 × 10-5 s-1 for the hydrolysis of the copper-bound probe. The probe was then validated in living mice using the copper chelator ATN-224 as a negative control, demonstrating that it can be used to monitor copper ions in vivo.