Functionalization of the meso‐Phenyl Ring of Rhodamine Dyes Through SNAr with Sulfur Nucleophiles: Synthesis, Biophysical Characterizations, and Comprehensive NMR Analysis

Abstract

AbstractAromatic nucleophilic substitution (SNAr) of fluorine in 9‐(3′‐carboxy‐4′,5′,6′,7′‐tetrafluorophenyl) groups of xanthene dyes constitutes a powerful tool in dye design. Thiols and amines regioselectively replace F‐6′. This approach enables additional hydrophilic residues or functional groups required for bioconjugation to be introduced. By using this methodology, a “bright” and photostable dye for two‐color superresolution microscopy was synthesized (with absorption and emission maxima at 604 and 627 nm, respectively). In the case of red‐emitting rhodamine dyes with 3′‐carboxy‐4′,5′,7′‐trifluorophenyl residues, two‐dimensional NMR techniques and a chemical transformation were used to prove the precise position of the additional substituent – a carboxylic acid group linked through the S‐atom at C‐6′. Furthermore, simple 1H NMR spectra reliably permit the position of the additional carboxy substituent in the 3′‐carboxyphenyl ring (at C‐5′ or C‐6′) to be established. Information on the exact position of this substituent is significant for the design of molecular probes and for the prediction of the properties of their bioconjugates.