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.

Highly Twisted N,N-Dialkylamines as a Design Strategy to Tune Simple Aromatic Hydrocarbons as Steric Environment-Sensitive Fluorophores

“…It is surprising and exciting that such primitive aromatic amines as BDAAs can achieve a large Stokes shift comparable to highly designed donor-acceptor systems. [44][45]50 …”

Super-resolution fluorescent materials: an insight into design and bioimaging applications

“…Moreover, another red-emitting dye KK114 possessing high photostability and good aqueous solubility has also been applied in STED microscopy successfully. 8 1 was first prepared by the conjugation of the 1-(3-carboxypropyl)-4-hydroxymethyl-2,2-dimethyl-1,2-dihydroquinoline fragment to the coumarin fluorophore with the 3-[2-(2-pyridyl)ethenyl] residue to implicate the absorption and emission bands at 472 and 623 nm, respectively. To circumvent this, Wurum et al furnished a series of rhodamine-based novel red fluorescent dyes possessing larger quantum yields with improved NHS ester stability (Fig.…”

Super-Resolution Molecular and Functional Imaging of Nanoscale Architectures in Life and Materials Science

“…Organic fluorophores suitable for STED microscopy have been identified by analyzing the energy level of the higher excited-state of the fluorophore (Hotta et al, 2010). Bright and photostable organic fluorophores with appropriate Stokes shifts have been designed for multicolor STED microscopy (Kolmakov et al, 2012; Schill et al, 2013). The photostability of the fluorophores is similarly one of the critical factors in SSIM.…”