title: 4-Trifluoromethyl-substituted coumarins with large Stokes shifts: synthesis, bioconjugates, and their use in super-resolution fluorescence microscopy.
abstract: Bright and photostable fluorescent dyes with large Stokes shifts are widely used as sensors, molecular probes, and light-emitting markers in chemistry, life sciences, and optical microscopy. In this study, new 7-dialkylamino-4-trifluoromethylcoumarins have been designed for use in bioconjugation reactions and optical microscopy. Their synthesis was based on the Stille reaction of 3-chloro-4-trifluoromethylcoumarins and available (hetero)aryl- or (hetero)arylethenyltin derivatives. Alternatively, the acylation of 2-trifluoroacetyl-5-dialkylaminophenols with available (hetero)aryl- or (hetero)arylethenylacetic acids followed by intramolecular condensation afforded coumarins with 3-(hetero)aryl or 3-[2-(hetero)aryl]ethenyl groups. Hydrophilic properties were provided by the introduction of a sulfonic acid residue or by phosphorylation of a primary hydroxy group attached at C-4 of the 2,2,4-trimethyl-1,2-dihydroquinoline fragment fused to the coumarin fluorophore. For use in immunolabeling procedures, the dyes were decorated with an (activated) carboxy group. The positions of the absorption and emission maxima vary in the ranges 413-480 and 527-668 nm, respectively. The phosphorylated dye, 9,CH=CH-2-py,H, with the 1-(3-carboxypropyl)-4-hydroxymethyl-2,2-dimethyl-1,2-dihydroquinoline fragment fused to the coumarin fluorophore bearing the 3-[2-(2-pyridyl)ethenyl] residue (absorption and emission maxima at 472 and 623 nm, respectively) was used in super-resolution light microscopy with stimulated emission depletion and provided an optical resolution better than 70 nm with a low background signal. As a result of their large Stokes shifts, good fluorescence quantum yields, and adequate photostabilities, phosphorylated coumarins enable two-color imaging (using several excitation sources and a single depletion laser) to be combined with subdiffractional optical resolution.
authors.affiliation: Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen (Germany), Fax: (+49) 551-2012505.
authors.forename: Heiko
authors.initials: H
authors.lastname: Schill
authors.forename: Shamil
authors.initials: S
authors.lastname: Nizamov
authors.forename: Francesca
authors.initials: F
authors.lastname: Bottanelli
authors.forename: Jakob
authors.initials: J
authors.lastname: Bierwagen
authors.forename: Vladimir N
authors.initials: VN
authors.lastname: Belov
authors.forename: Stefan W
authors.initials: SW
authors.lastname: Hell
mesh_terms: D003374:Coumarins / Q000138:chemical synthesis / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D054879:Halogenation; D006367:HeLa Cells; D006801:Humans; D008745:Methylation; D008856:Microscopy, Fluorescence
chemical_list: D003374:Coumarins; D005456:Fluorescent Dyes; C030123:coumarin
keywords: bioconjugation; chromophores; fluorescence; imaging agents; light microscopy