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

Abstract

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.

Vibrationally Resolved Absorption, Fluorescence, and Preresonance Raman Spectroscopy of Isolated Pyronin Y Cation at 5 K

“…This can occur when alkyl-substituted aryl-amino groups (e.g., the dimethyl amine group in [PY] + ) rotate out of conjugation with the π-system. 18,40 [PY] • appears to undergo an analogous twisting deformation upon photoexcitation. The phenomenon of a dark or weakly emitting neutral and strongly fluorescent cation is surprisingly rare among molecular chromophores.…”

“…It is a well-known, bright green-yellow fluorophore and the most commonly utilized fluorescent probe among the pyronin family ([PY] + , pyronin B, and acridine red) . Since its first synthesis in 1894, , the fluorescent and selective RNA staining properties of pyronin Y and its synthetically tailored derivatives have been exploited in many applications, e.g., in histochemistry, , optoelectronics, , and microscopy. , As a result, pyronin Y has also been the subject of many experimental and theoretical studies to better understand its spectral properties, e.g., toward improving fluorescence quantum yields (Φ f ) and enhancing molar absorptivity by appropriate functionalization. − Despite this intense activity, a molecular-level description of the vibronic properties of this model fluorophore with high spectroscopic accuracy is still missing. This reflects the inherent difficulty of selectively preparing well-defined samples of charged chromophores for spectroscopy.…”

“…A well-known mechanism leading to a reduced Φ f of many organic fluorophores in the condensed phase is twisted intermolecular charge transfer (TICT). This can occur when alkyl-substituted aryl-amino groups (e.g., the dimethyl amine group in [PY] + ) rotate out of conjugation with the π-system. , [PY] • appears to undergo an analogous twisting deformation upon photoexcitation.…”