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

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 pi-conjugation. In the spectroscopic measurements and computational calculations, enamine luciferin showed more red-shifted absorption and fluorescence emission than LH2 and iLH(2). 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.

Automated summary

A new synthetic luciferin, designed with rotationally restricted structure, demonstrated enhanced performance for bioluminescence reaction. This luciferin possessed two intramolecular H-bonds to limit degrees of freedom, an amine group to promote indirect light conversion, and an alkenyl group to increase π-conjugation. Spectroscopic and computational studies showed that the synthetic luciferin exhibited longer absorption and fluorescence emission wavelength compared to LH2 and iLH(2). Docking studies suggested that the emission wavelength of the luciferin might be influenced by the twisted structure caused by Asp531 within the enzyme. Further investigation with mutant luciferase FlucRed revealed a major emission peak at 606 nm, a distinct shoulder above 700 nm, and 21% of the spectrum located in the near-infrared range.