Compared to DTZ, 8pyDTZ (compounds 3c) is able to emit red-shifted chemiluminescence or bioluminescence. 8pyDTZ shows the most red-shifted emission and red-shifted photons can penetrate through tissue better.
LumiLuc-8pyDTZ in human embryonic kidney (HEK) 293T cells transiently expressing the luciferase is evaluated. The LumiLuc-8pyDTZ pair produces ～3-5-fold more bioluminescence than teLuc-8pyDTZ. To compare far-red emission intensities of bioluminescent reporters, HEK 293T cells in the presence of a 600-700 nm band pass filter are imaged. At substrate concentrations from 6.25 to 100 μM, LumiLuc-8pyDTZ consistently produces 1.6-3.9-fold higher photon flux than teLuc-DTZ. No ATP perturbation is observed from 8pyDTZ-treated, LumiLucexpressing cells.

LumiLuc-8pyDTZ pair is compared with several benchmark reporters in a tumor xenograft mouse model. LumiLuc-8pyDTZ shows high sensitivity and does not need organic cosolvents for in vivo administration. LumiScarlet-8pyDTZ is comparable to Akaluc-AkaLumine, the brightest ATP-dependent luciferase-luciferin pair, for detecting cells in deep tissues of mice.