Bioluminescence, photophysical, computational and molecular docking studies of fully conformationally restricted enamine infraluciferin

Abstract

A new rationally designed fully rotationally restricted luciferin has been synthesised. This synthetic luciferin, based upon the structure of infraluciferin, has two intramolecular H-bonds to reduce degrees of freedom, an amine group to enhance ICT process, and an alkenyl group to increase π-conjugation. In the spectroscopic measurements and computational calculations, enamine luciferin showed more red-shifted absorption and fluorescence emission than LH₂ and iLH₂. With PpyWT luciferase enamine luciferin gave bioluminescence at 564 nm which is similar to LH₂ at 561 nm. Further investigation by docking studies revealed that the emission wavelength of enamine luciferin might be attributed to the unwanted twisted structure caused by Asp531 within the enzyme. With mutant luciferase FlucRed, the major emission peak was shifted to 606 nm, a distinct shoulder above 700 nm, and 21% of its spectrum located in the nIR range.