SYNTHESIS OF PROBE MOLECULES, 6-(DIMETHYLAMINO)-2-PHENYLISOINDOLIN-1-ONES, FOR MECHANISTIC STUDIES OF FIREFLY LUCIFERASE INHIBITION.

Firefly luciferase is used in high-throughput screening based on the detection of chemiluminescence. It catalyzes an esterification reaction of luciferin with adenosine 5'-triphosphate (ATP) followed by decarbonylation with oxygen and concomitance of light. Previously, we reported that firefly luciferase also possesses acyl-CoA synthetase activity and catalyzes an aromatic carboxylic acid group of F-53, using ATP, Mg and coenzyme A (CoA), to produce F-53 covalently attached to active-site lysine-529 residue of firefly luciferase through the formation of an amide group. The amidation of lysine-529 resulted in a deactivation of luciferase. In order to probe firefly luciferase inhibition's mechanism, we synthesized two probe molecules and , mimicking F-53. Molecule contains an azido-appended side chain in the aromatic ring of F-53, while possesses an azido and a carboxylic acid group appended side chains. Both synthetic schemes are readily amenable to large-scale syntheses. Molecule was made from 2-allylaniline, which was derived from a thermal-induced aromatic-Claisen rearrangement of -allylaniline. The azido-appended side chain of was installed from a Horner-Wadsworth-Emmons reaction and the carboxylic acid side chain from a Sonogashira reaction.