Rhodamines and structurally similar rosamines are some of the most highly utilized tools for molecular imaging experiments. We report a general and high-yielding route to produce 18 examples of rhodamines and rosamines, including tetramethylrhodamine, rhodamine B, and Janelia Fluor 549, from a single xanthone intermediate, 3,6-difluoroxanthone. Spectroscopic studies revealed trends in fluorophore efficiency based on substitution patterns at the 3′-, 6′-, and 9′-positions, providing insights to aid future designs of rhodamines/rosamines.

The pyronin class of fluorophores serves a critical role in numerous imaging applications, particularly involving preferential staining of RNA through base pair intercalation. Despite this important role in molecular staining applications, the same set of century-old pyronins (i.e., pyronin Y (PY) and pyronin B (PB)), which possess relatively low fluorophore brightness, are still predominantly being used due to the lack of methodology for generating enhanced variants. Here, we use TD-DFT calculations of interconversion energies between structures on the S 1 surface as a preliminary means to evaluate fluorophore brightness for a proposed set of pyronins containing variable substitution patterns at the 2, 3, 6, and 7 positions. Using a nucleophilic aromatic substitution/hydride addition approach, we synthesized the same set of pyronins and demonstrate that quantum-mechanical computations are useful for predicting fluorophore performance. We produced the brightest series of pyronin fluorophores described to date, which possess considerable gains over PY and PB.