Fluorescently labeled R848, R837, and SM360320 are TLR7 agonists. R848 (A), R837 (B), and SM (C) were conjugated with TAMRA (in red) or with Alexa Fluor 488 (AF488; in green) fluorophores at the indicated tolerant linker site. A spacer was introduced together with TAMRA (in orange). Both fluorophores were a mixture of 5-6 regioisomers. Structures depicted here represent the 5-regioisomer. The biologic activity of these fluorescently labeled molecules was assessed on HEK293T cells stably transfected with TLR7 and the luciferase gene under the transcriptional control of NF-κB and (TLR7-DST) in parallel with their nonfluorescent parent molecules. Luciferase activity was expressed as fold induction (FI) over nonstimulated cells. EC₅₀ values are reported.