Redox-active metals have long been recognized as essential micronutrients and their dysregulation has been associated with various disease pathologies including cancer. As such, the ability to visualize the movement of these metal pools would provide key insight into mechanisms underlying cancer progression. However, current techniques for longitudinal monitoring of redox-active metals in vivo are lacking and limited in their selectivity, sensitivity, and spatial control. Bioluminescent imaging systems use a substrate (luciferin) paired to a luciferase enzyme to selectively generate photons in the presence of an analyte. This is typically done by chemically modifying the luciferin, termed “caging” so that it is unable to interact with it’s luciferase pair and generate light unless it is in the presence of an analyte. We are developing a bioluminescent imaging probe for monitoring extracellular copper pools. Bioluminescent probes provide numerous advantages including high sensitivity and targeted localization. In this presentation, we describe the synthesis and characterization of three caged substrates, all containing the coelenterazine-type imidazopyrazine core. The parent cores of our three caged luciferins are compatible with marine luciferases which do not require ATP as a cofactor unlike the traditional firefly luciferase, making them good candidates for extracellular imaging. We demonstrate that these probes exhibit copper-selective luminescence in the presence of the recombinant marine luciferase, Gaussia luciferase, and an artificial derivative, Nanoluciferase, as well as in cell lines secreting those luciferases.

Development of bioluminescent probes for visualizing extracellular, labile copper

ACS Spring 2022

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