Red-shifted d-luciferin analogues and their bioluminescence characteristics

Abstract

D-Luciferin (D-LH₂) is the most used substrate for beetle luciferases in various bioluminescence applications. Here, we successfully synthesized six D-LH₂ analogues including 5′,7′-dimethoxy-D-LH₂ and 7′-methylnaphthol-D-LH₂ as novel compounds. We also developed a continuous one-pot green synthesis method to improve yields of luciferins from condensation of quinone and D-Cys (63-fold greater than the previous report). The novel D-LH₂ analogues were tested with five luciferases (Fluc, SLR, Eluc, Pmluc-WT, and Pmluc-N230S), and all the compounds emitted bioluminescence at wavelengths longer than that of D-LH₂ (>80 nm). The reaction of SLR with 5′,7′-dimethoxy-D-LH₂ gave the longest red-shifted bioluminescence at 663 nm. Remarkably, the reactions of 5′-methyl-D-LH₂ emit longer wavelengths and brighter light than those of D-LH₂ in all tested luciferases, except for Eluc. Interestingly, the novel red-shifted 5′,7′-dimethyl-D-LH₂ also provided prolonged bioluminescence with a rate of light decay slower than that of D-LH₂. We further demonstrated applications of 5′-methyl-D-LH₂ and 5′,7′-dimethyl-D-LH₂ in mammalian cell lines expressing Fluc, SLR, and Pmluc-N230S. 5′-Methyl-D-LH₂ provided about 11.2-fold greater sensitivity to detect Fluc in the HEK293T crude lysate than D-LH₂, achieving the detection with a lower number of cell lines. The red-shifted 5′,7′-dimethyl-D-LH₂ also exhibits high sensitivity when using a red light filter to monitor live cell bioluminescence. These D-LH₂ analogues, 5′-methyl-D-LH₂ and 5′,7′-dimethyl-D-LH₂, are promising substrates for future cell-based assays and real-time monitoring applications.