Red-shifted <scp>d</scp> -luciferin analogues and their bioluminescence characteristics

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.