Synthesis and bioluminescence of electronically modified and rotationally restricted colour-shifting infraluciferin analogues

Synthetic nIR emitting luciferins can enable clearer bioluminescent imaging in blood and tissue. A limiting factor for all synthetic luciferins is their reduced light output with respect to D-luciferin. In this work we explore a design feature of whether rigidification of an exceptionally red synthetic luciferin, infraluciferin, can increase light output through a reduction in the degrees of freedom of the molecule. A rigid analogue pyridobenzimidazole infraluciferin was prepared and its bioluminescence properties compared with its non-rigid counterpart benzimidazole infraluciferin, luciferin, infraluciferin and benzimidazole luciferin. The results support the concept that synthetic rigidification of π-extended luciferins can increase bioluminescence activity while maintaining nIR bioluminescence.