Aryliden-Imidazolones as Fluorogens of NanoLuc Protein

Objective: Fluorogens are synthetic molecules that become fluorescent in specific environments, i.e. in pockets of fluorogen-activating protein (FAP). In pursuit of increased diversity of fluorescent labeling methods, our group is interested in developing new FAP-fluorogen pairs with unique features. Methods: In this study, we focused on synthesis of prospective fluorogens for NanoLuc luciferase used as FAP. A row of orto-substituted aryliden-imidazolones and their derivatives containing styrene moiety was synthesized and tested in vitro with purified NanoLuc protein. The selected dye was used as fluorogen in widefield microscopy of live HEK 293 cells transiently transfected with NanoLuc-H2B coding plasmid. Results and Discussion: The structure of the GFP and Kaede proteins chromophores is very similar to the structure of the luciferins of the NanoLuc luciferase. In this regards, we synthesized a series of such arylidene-imidazolones and tested them with NanoLuc protein. It was found that some the created compounds are able to bind to the NanoLuc protein and form fluorescent complexes. One of them had a high affinity for NanoLuc protein and was used as a successful fluorogen in pair with NanoLuc in live cell microscopy. Conclusions: We report a series of orto-substituted aryliden-imidazolones and their derivatives containing styrene moiety. These compounds can be used as ligands of NanoLuc protein. Together with NanoLuc these fluorogens can be used for genetically encoded labeling in fluorescence microscopy, as demonstrated by staining HEK293 cells.