title: Uptake, distribution and diffusivity of reactive fluorophores in cells: implications toward target identification.
abstract: There is much recent interest in the application of copper-free click chemistry to study a wide range of biological events in vivo and in vitro. Specifically, azide-conjugated fluorescent probes can be used to identify targets which have been modified with bioorthogonal reactive groups. For intracellular applications of this chemistry, the structural and physicochemical properties of the fluorescent azide become increasingly important. Ideal fluorophores should extensively accumulate within cells, have even intracellular distribution, and be free (unbound), allowing them to efficiently participate in bimolecular reactions. We report here on the synthesis and evaluation of a set of structurally diverse fluorescent probes to examine their potential usefulness in intracellular click reactions. Total cellular uptake and intracellular distribution profiles were comparatively assessed using both quantitative and qualitative approaches. The intracellular diffusion coefficients were measured using a fluorescence recovery after photobleaching (FRAP)-based method. Many reactive fluorophores exhibited suboptimal properties for intracellular reactions. BODIPY- and TAMRA-based azides had superior cellular accumulation, whereas TAMRA-based probes had the most uniform intracellular distribution and best cytosolic diffusivity. Collectively, these results provide an unbiased comparative evaluation regarding the suitability of azide-linked fluorophores for intracellular click reactions.
authors.affiliation: Department of Medicinal Chemistry and Pharmaceutical Chemistry, Molecular Graphics and Modeling Laboratory, and Specialized Chemistry Center, University of Kansas, Lawrence, Kansas, USA.
authors.forename: Christopher W
authors.initials: CW
authors.lastname: Cunningham
authors.forename: Archana
authors.initials: A
authors.lastname: Mukhopadhyay
authors.forename: Gerald H
authors.initials: GH
authors.lastname: Lushington
authors.forename: Brian S J
authors.initials: BS
authors.lastname: Blagg
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Prisinzano
authors.forename: Jeffrey P
authors.initials: JP
authors.lastname: Krise
mesh_terms: D001386:Azides / Q000737:chemistry; D002460:Cell Line; D036681:Fluorescence Recovery After Photobleaching; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D008856:Microscopy, Fluorescence; D015394:Molecular Structure; D012235:Rhodamines / Q000737:chemistry
chemical_list: D001386:Azides; D005456:Fluorescent Dyes; D012235:Rhodamines; C005358:tetramethylrhodamine