New Properties of a Bioinspired Pyridine Benzimidazole Compound as a Novel Differential Staining Agent for Endoplasmic Reticulum and Golgi Apparatus in Fluorescence Live Cell Imaging

In this study, we explored new properties of the bioinspired pyridine benzimidazole compound (2,4-di-tert-butyl-6-(3H-imidazo[4,5-c]pyridine-2-yl)phenol) regarding its potential use as a differential biomarker. For that, we performed 1D HNMR (TOCSY), UV-Vis absorption spectra in different organic solvents, voltammetry profile (including a scan-rate study), and TD-DFT calculations that including NBO analyses, to provide valuable information about structure and luminescence. In our study, we found that the structure is highly stable, where the presence of an intramolecular hydrogen bond (IHB) seems to have a crucial role in the stability of luminescence, and its emission can be assigned as fluorescence. In fact, we found that the relatively large Stokes Shift observed for (around 175 nm) may be attributed to the stability of the geometry and the strength of its IHB. On the other hand, we determined that is biocompatible by cytotoxicity experiments in HeLa cells, an epithelial cell line. Furthermore, in cellular assays we found that could be internalized by passive diffusion in absence of artificial permeabilization at short incubation times (15 min to 30 min). Fluorescence microscopy studies confirmed that accumulates in the endoplasmic reticulum (ER) and Golgi apparatus, two organelles involved in the secretory pathway. Finally, we determined that exhibited no noticeable blinking or bleaching after 1 h of continuous exposure. Thus, provides a biocompatible, rapid, simple, and efficient way to fluorescently label particular organelles, producing similar results to that obtained with other well-established but more complex methods.