Improving the Brightness of Pyronin Fluorophore Systems through Quantum-Mechanical Predictions

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 S₁ 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.