Bioluminescence imaging with luciferin-luciferase pairs has been widely used in the biomedical research field. One of the most commonly used luciferin-luciferase pairs is the firefly bioluminescence system. In order to apply firefly bioluminescence as biological reporter, various firefly luciferin analogues have been synthetically developed, so far. Among them, the amino luciferin, in which the hydroxyl group at C6` position of D-luciferin was replaced by an amino group, has been extensively studied based on its bioluminescence activity and applicability. Because the chemical structure of the luciferin strongly affects the bioluminescence output, methods for emission colour and brightness modulation are often similar to those in development of fluorophores. For example, electron donating substituents and rigidity at the C6` position of firefly luciferin strongly affect the π-electronic system and spectroscopic properties.

In 2015, Lavis et al. established a general structural modification that improves the brightness (fluorescence quantum yield) of fluorophores. In particular, a minor alteration of an N,N-dimethylamino substituent in a classical dye with a cyclic amine, especially azetidine, greatly improved the fluorescence quantum efficiency by suppressing twisted internal charge transfer.

To develop multicolor, high brightness and functional firefly luciferins, we designed and developed firefly luciferins incorporating azacyclic amine rings at the C6` position. With azacyclic analogues in hand, bioluminescence spectra were evaluated with native firefly luciferase. All azacyclic analogues exhibited bioluminescence emission with slight red shift. We next evaluated the bioluminescence quantum yield of the analogues. The quantum yield of a bioluminescence reaction is defined as the efficiency of a photon production from a single reactant luciferin. Azacyclic luciferins showed improved quantum yield compared with non-cyclic amino analogues, presumably due to the improvement of the fluorescence quantum yield by the ring structure. Moreover, evaluation of kinetic parameters revealed that the azacyclic ring structure dramatically improved the affinity to native luciferase.