Methylation-controlled photophysical tuning in isolated acridine cations revealed by cryogenic fluorescence spectroscopy and TD-DFT

Abstract

Acridine derivatives, such as proflavine, acriflavine, and acridine orange, have been used extensively in biology and biomedicine as fluorescent probes by forming DNA-intercalating complexes. This approach benefits from a comprehensive understanding of their photophysical properties. In this context, we studied the fluorescence properties of proflavine, a reference compound, and its methylated derivatives under cryogenic, isolated conditions. Fluorescence excitation and dispersed emission spectra were measured, and spectral interpretation was supported by time-dependent density functional theory (TD-DFT) calculations at the ωB97XD/aug-cc-pVDZ level of theory. The excitation and emission spectra exhibit progressively red-shifted maxima with increasing methylation, reaching shifts up to 0.270 eV in emission, while the vibronic structure evolves from sharp to broad depending on the methylation site. Molecular geometry optimization, in both ground and excited states, predicts that the methylation of the amino side groups maintains the planar geometry observed in proflavine. In contrast, methylation of the nitrogen heterocycle disrupts symmetry, leading to an out-of-plane bend. These geometric differences lead to distinct active vibronic modes in Franck–Condon simulations, providing an explanation for the observed spectral differences. Additionally, TD-DFT calculations reproduce the red-shift trend experimentally observed, although they systematically overestimate excitation energies. Overall, the findings establish methylation as an effective strategy for tuning the photophysics of diaminoacridines, demonstrating that side substitution enables controlled spectral shifts without compromising emission efficiency or spectral resolution. These insights provide a rational framework for designing functional dyes with tailored optical properties for biological applications.