Expedient Synthesis of Electronically Modified Luciferins for Bioluminescence Imaging

Bioluminescence imaging with luciferase enzymes requires access to light-emitting, small-molecule luciferins. Here, we describe a rapid method to synthesize d-luciferin, the substrate for firefly luciferase (Fluc), along with a novel set of electronically modified analogues. Our procedure utilizes a relatively rare, but synthetically useful dithiazolium reagent to generate heteroaromatic scaffolds in a divergent fashion. Two of the luciferin analogues produced with this approach emit light with Fluc <i>in vitro</i> and in live cells. Collectively, our work increases the number of substrates that can be used for bioluminescence imaging and provides a general strategy for synthesizing new collections of luciferins