Anthracene-Pyridine Derivatives as Fluorescent Probes: The Role of Nitrogen Positioning in Bioimaging Performance

Abstract

The strategic design of small-molecule fluorescent probes is critical for advancing precision bioimaging in both cellular and microbiological contexts. In this study, we report a series of anthracene-pyridine derivatives-compounds 3a, 3b, and 3c-with nitrogen atoms positioned at the ortho, meta, and para positions, respectively, to investigate how atomic-level substitution patterns govern photophysical properties and imaging performance. Spectroscopic characterization revealed that the para-substituted compound 3c exhibits enhanced intramolecular charge transfer (ICT) character, leading to lower fluorescence quantum yield in polar biological environments due to increased non-radiative decay. In contrast, the meta-substituted derivative 3b maintains a more locally excited (LE)-like emission, producing intense blue fluorescence and demonstrating high selectivity for bacterial imaging, likely due to favourable interactions with nucleic acids or membranes. Ortho-substituted compound 3a also displayed appreciable fluorescence in mammalian cells, though with lower intensity and reduced bacterial uptake. Complementary molecular dynamics simulations revealed that nitrogen positioning influences molecular geometry, conformational stability, and interaction propensities with biological targets, thereby shaping the observed bioimaging performance. Collectively, these findings establish clear structure-property-function relationships, underscoring how fine-tuning nitrogen placement can optimize excited-state behaviour, cellular uptake, and emission output. These insights provide a valuable framework for the rational design of next-generation fluorophores tailored for multicolour and environment-sensitive imaging applications.