Bioluminescence (BL) is widely used as an optical readout in bioassays and molecular imaging applications. In this study, we present a systematic approach to develop an advanced multiplex imaging system using novel coelenterazine (CTZ) analogs, termed the "G-series." We synthesized a library of 20 CTZ analogs with diverse functional groups at the C-2 and C-8 positions of the imidazopyrazinone backbone and classified them into five categories. These analogs were characterized with respect to luciferase specificity, BL stability, emission spectra, kinetics, and computationally predicted binding modes. Most of the G-series CTZ analogs were found to be water-soluble and exhibited excellent specificity for NanoLuc from Oplophorus luciferase, while
Kamiya et al. (Fri,) studied this question.