The authors were able to synthesize luciferins with benzimidazole and imidazoline rings. Benzimidazoles are known competitive inhibitors of Fluc, and previous work has shown that such heterocycles can be excited to emit light. After obtaining these nitrogenous luciferins, these compounds were used as substrates for Fluc in the presence of ATP and CoASH. No bioluminescence could be detected for analog 3 (see Figure), and only a very weak signal was obtained for analog 4. However, analogs 2 and 15 showed detectable signals, although they were approximately 100-fold weaker than the one obtained with D-luciferin. The benzimidazole analog 2 showed a red-shifted emission maximum of 578 nm.
Interestingly, analog 15 showed a greatly blue-shifted emission peak compared to other luciferins with a λmax = 460 nm. This emission is similar to what is found with coelenterazine if used as a substrate for the luciferase isolated from Renilla reniformis. These analogs were also shown to function in a cell-based Fluc assay using HEK293 cells.
Studies such as these should extend the range of available substrates for Fluc providing researchers new formulations for use in both in vitro and in vivo studies.