An easily synthesized, photolyzable luciferase substrate for in vivo luciferase activity measurement

Many reporter gene assays require killing the cell by fixation or lysis. For assays in living cells, the substrate delivery is inefficient and cannot be supplied in situ in a bolus, which makes assays highly variable. We report a simple synthesis of a luciferin ester that is both photolyzable and cleaved by endogenous esterases such that luciferase activity in living cells is easily monitored. Although the photolyzed substrate can be delivered in bolus, the rapid equilibration of the luciferin ester in the cell and the continuous delivery by the endogenous esterases allow stable, long-term measurements of luciferase activity.