Watthaisong P.Kantiwiriyawanitch C.Jitkaroon W.Phintha A.Klayparn I.Lawan N.Kamutira P.Sasaki D.Visitsatthawong S.Maenpuen S.Tinikul R.Sucharitakul J.Nishihara R.Niwa K.Nakajima Y.Ohmiya Y.Chaiyen P.Mahidol University2025-12-082025-12-082025-01-01Rsc Chemical Biology (2025)https://repository.li.mahidol.ac.th/handle/123456789/113437d-Luciferin (d-LH<inf>2</inf>) is the most used substrate for beetle luciferases in various bioluminescence applications. Here, we successfully synthesized six d-LH<inf>2</inf> analogues including 5′,7′-dimethoxy-d-LH<inf>2</inf> and 7′-methylnaphthol-d-LH<inf>2</inf> 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-LH<inf>2</inf> 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-LH<inf>2</inf> (>80 nm). The reaction of SLR with 5′,7′-dimethoxy-d-LH<inf>2</inf> gave the longest red-shifted bioluminescence at 663 nm. Remarkably, the reactions of 5′-methyl-d-LH<inf>2</inf> emit longer wavelengths and brighter light than those of d-LH<inf>2</inf> in all tested luciferases, except for Eluc. Interestingly, the novel red-shifted 5′,7′-dimethyl-d-LH<inf>2</inf> also provided prolonged bioluminescence with a rate of light decay slower than that of d-LH<inf>2</inf>. We further demonstrated applications of 5′-methyl-d-LH<inf>2</inf> and 5′,7′-dimethyl-d-LH<inf>2</inf> in mammalian cell lines expressing Fluc, SLR, and Pmluc-N230S. 5′-Methyl-d-LH<inf>2</inf> provided about 11.2-fold greater sensitivity to detect Fluc in the HEK293T crude lysate than d-LH<inf>2</inf>, achieving the detection with a lower number of cell lines. The red-shifted 5′,7′-dimethyl-d-LH<inf>2</inf> also exhibits high sensitivity when using a red light filter to monitor live cell bioluminescence. These d-LH<inf>2</inf> analogues, 5′-methyl-d-LH<inf>2</inf> and 5′,7′-dimethyl-d-LH<inf>2</inf>, are promising substrates for future cell-based assays and real-time monitoring applications.ChemistryBiochemistry, Genetics and Molecular BiologyArticleSCOPUS10.1039/d5cb00287g2-s2.0-10502342021026330679