Divergent Synthesis of Ultrabright and Dendritic Xanthenes for Enhanced Click-Chemistry-Based Bioimaging

Biorthogonal labelling with fluorescent small molecules is an indispensable tool for diagnostic and biomedical applications. In dye-based 5-ethynyl-2'-deoxyuridine (EdU) cell proliferation assays, augmentation of the fluorescent signal entails an overall enhancement in the sensitivity and quality of the method. To this end, a rapid, divergent synthetic procedure that provides ready-to-click pH-insensitive rhodamine dyes exhibiting outstanding brightness was established. Compared to the shortest available synthesis of related high quantum-yielding rhodamines, two fewer synthetic steps are required. In a head-to-head imaging comparison involving copper(I)-catalyzed azide alkyne cycloaddition reactions with in vitro administered EdU, our new 3,3-difluoroazetidine rhodamine azide outperformed the popular 5-TAMRA-azide, making it among the best available choices when it comes to fluorescent imaging of DNA. In a further exploration of the fluorescence properties of these dyes, a set of bis-MPA dendrons carrying multiple fluorescein or rhodamine units was prepared by branching click chemistry. Fluorescence self-quenching of fluorescein- and rhodamine-functionalized dendrons limited the suitability of the dyes as labels in EdU-based experiments but provided new insights into these effects.