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

Graphical Abstract

Aromatic nucleophilic substitution of fluorine in 9-(3′-carboxy-4′,5′,6′,7′-trifluorophenyl) groups of xanthene dyes is regioselective (for thiols and amines). The reaction was used for the synthesis of a “bright” and very photostable dye for two-color superresolution microscopy. Characteristics of the NMR spectra may be used for structure elucidation of other fluorescent dyes.

Abstract

Aromatic nucleophilic substitution (S_NAr) 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.