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Paper | Special issue | Vol 103, No. 1, 2021, pp.231-248
Published online, 17th April, 2020
DOI: 10.3987/COM-20-S(K)1
■ Synthesis of Probe Molecules, 6-(Dimethylamino)-2-phenylisoindolin-1-ones, for Mechanistic Studies of Firefly Luciferase Inhibition

Medha J. Gunaratna, Bo Hao, Man Zhang, Madoka Nakagomi, Ai Ito, Takeo Iwamoto, and Duy H. Hua*

*Department of Chemistry, Kansas State University, Manhattan, KS 66506, U.S.A.

Abstract

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, Mg2+ 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 1 and 2, mimicking F-53. Molecule 1 contains an azido-appended side chain in the aromatic ring of F-53, while 2 possesses an azido and a carboxylic acid group appended side chains. Both synthetic schemes are readily amenable to large-scale syntheses. Molecule 1 was made from 2-allylaniline, which was derived from a thermal-induced aromatic-Claisen rearrangement of N-allylaniline. The azido-appended side chain of 2 was installed from a Horner-Wadsworth-Emmons reaction and the carboxylic acid side chain from a Sonogashira reaction.