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

Aromatic 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 H-1 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.