Designing anthracene-based D-π-a fluorophores with tunable excited-state dynamics for cellular and bacterial imaging.

Abstract

The rational design of donor-π-acceptor (D-π-A) fluorophores requires an understanding of how molecular structure governs not only steady-state optical properties but also excited-state relaxation and emission dynamics in different environments. Here, we systematically investigate three triphenylamine-anthracene fluorophores bearing a cyano-substituted styryl-pyridyl unit (TA-AnStCNPy), a styryl-pyridyl unit (TA-AnStPy), or a phenyl-pyridyl unit (TA-ANPy), to elucidate the influence of cyano functionalization and conjugated-linker modification on their photophysical behaviour. By combining steady-state absorption and fluorescence spectroscopy, density functional theory (DFT/TD-DFT) calculations, and wavelength-resolved time-resolved photoluminescence, we establish distinct relationships between molecular architecture, solvent-dependent emission, and condensed-phase excited-state dynamics. Under common solvent conditions, TA-AnStCNPy exhibits the most red-shifted fluorescence, while TA-AnStPy displays pronounced solvatochromism across an extended seven-solvent series, with emission maxima shifting from 439 to 519 nm despite relatively unchanged absorption maxima (394-399 nm). Time-resolved measurements reveal further differences that cannot be inferred from steady-state spectroscopy alone. TA-AnStCNPy exhibits substantial time-dependent spectral relaxation in neat films, which is markedly reduced upon dilution in a Zeonex polymer matrix. In contrast, TA-AnStPy shows the most pronounced excitation-fluence-dependent long-lived emission, accompanied by a spectrally distinct red-shifted component, whereas TA-ANPy remains predominantly prompt-emissive. These observations highlight the different sensitivities of excited-state relaxation and long-lived emission to molecular structure and the surrounding medium, without establishing a unique triplet-mediated mechanism. Complementary fluorescence microscopy demonstrates detectable fluorescence associated with HDFa cells and Escherichia coli preparations, while MTT assays indicate retained cellular metabolic activity at imaging-relevant concentrations. Collectively, this study demonstrates that structural modifications producing distinct solution-state optical responses can also lead to markedly different excited-state dynamics in condensed phases, providing a comparative framework for the development of anthracene-based fluorophores with tunable photophysical properties.

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