Chemi- and bio-luminescence of coelenterazine analogues with phenyl homologues at the C-2 position

Abstract

A series of phenyl homologues of coelenterazine substituted at the C-2 position were synthesized and their bio- and chemi-luminescence properties were investigated including the measurement of chemiluminescence spectra in various media. The light emitting species of each analogue was found to be a neutral form of a coelenteramide derivative in diethylene glycol dimethyl ether (DGM) containing a trace amount of acetate buffer (pH 5.60), while a monoanion was found only in dimethyl sulfoxide (DMSO) and a dianion was observed in DMSO containing a trace amount of aqueous sodium hydroxide. Based on pseudo first-order reaction kinetics, chemiluminescence rate constants were obtained in DGM containing a trace amount of acetate buffer. Each of the synthetic coelenterazine analogues was incorporated into recombinant apoaequorin to obtain a series of semi-synthetic aequorins. Measurements of bioluminescence activities of the aequorins revealed that a benzyl group in the C-2 position was essential for efficient luminescence activity. A two-step incubation procedure was used to determine why some analogues gave less luminescence activity than the benzyl analogue and natural coelenterazine.