Coumarin Luciferins and Mutant Luciferases for Robust Multicomponent Bioluminescence Imaging

We prepared a new class of luciferins based on a red-shifted coumarin scaffold. These probes (CouLuc-1s) were accessed in a two-step sequence via direct modification of commercial dyes. The bioluminescent properties of the CouLuc-1 analogs were also characterized, and complementary luciferase enzymes were identified using a two-pronged screening strategy. The optimized enzyme-substrate pairs displayed robust photon outputs and emitted a significant portion of near-infrared light. The CouLuc-1 scaffolds are also structurally distinct from existing probes, enabling rapid multicomponent imaging. Collectively, this work provides novel bioluminescent tools along with a blueprint for crafting additional probes for multiplexed imaging