Convergent synthesis and optical properties of near-infrared emitting bioluminescent infra-luciferins

Infra-luciferin, an alkene linked analogue of luciferin, gives bioluminescence emission >700 nm and has the potential to be used for multiparametric in vivo imaging. We report here a high yielding, scalable and convergent synthesis of infra-luciferin which will allow the synthesis of other conjugated luciferins for investigation in near-infrared bioluminescence imaging. We demonstrated this potential by using the new route to synthesise a diene linked analogue of luciferin, the fluorescent and bioluminescent properties of which were compared to those of d-luciferin and infra-luciferin. We found that extension of conjugation to a diene linker resulted in the specific bioluminescence activity being reduced by 3–4 orders of magnitude compared to d-luciferin. Analogous to its fluorescence emission spectrum, the diene linked analogue exhibited two peaks in its bioluminescence spectrum, the major one being slightly blue-shifted compared to natural d-luciferin, and a minor peak at ca. 800 nm. The fluorescence quantum yield and pH dependence of fluorescence were also determined.