Optical imaging technologies play important roles in the biomedical research field. Nowadays, bioluminescence has become a standard non-invasive modality to trace biological functions without having to sacrifice animals. The most commonly used luciferin-luciferase pairs in these assays originate from insects, such as firefly and click beetles. These bioluminescence systems consist of D-luciferin and the corresponding insect luciferases, for example Fluc from photinus pylalis, and emit yellow-green to orange light (560-630 nm). Although D-luciferin has been widely applied to in vitro and in vivo imaging, it is sometimes not the ideal substrate for in vivo imaging, especially in deep tissues such as mouse lung, liver or brain. The light emitted from D-luciferin suffers from strong light absorption by blood components and tissue, making in vivo imaging difficult. To address this drawback, several synthetic luciferins emitting in the near-infrared (NIR) region (650-900 nm) have been developed. However, there is still less variety in NIR luciferins compared to fluorescent probes. In this study, we have designed and synthesized a series of NIR synthetic luciferins via a ring fusion strategy, using AkaLumine, which is one of the NIR luciferins, as a framework. Fortunately, all synthetic luciferins were tolerated by Fluc, which is one of the native luciferases, and showed NIR emission up to 690 nm. Since the bioluminescence signal should reflect the amount of reporter luciferase, luciferins with higher luciferase affinity (low Km value) are desirable. Evaluation of enzymatic parameters revealed that the synthetic luciferins have higher affinity for Fluc, Therefore, this result indicates that a lower amount of luciferin is sufficient to saturate the luciferase compared to D-luciferin. Moreover, we applied the panel of synthetic luciferins to Fluc-expressing live cells in order to verify cell membrane permeability. As a result, all synthetic luciferins emit detectable light and their luminescence spectra were in agreement with the results obtained using purified luciferase.

Structure-luminescence relationship of near-infrared firefly luciferin analogues

2019 Fall National Meeting

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