A Fluorescent Probe for Protein Misfolding and Aggregation Due to Oxidative Stress Based on a 7-Azaindole-BODIPY Derivative

Abstract

The development of new fluorescent probes as molecular sensors is a critical step for the understanding of molecular mechanisms. Probes based on BODIPY offer remarkable versatility in molecular sensing due to their unique properties. BODIPY-based probes exhibit high fluorescence quantum yields, exceptional photostability, and tunable absorption/emission wavelengths. Here, we report the synthesis and evaluation of a novel 7-azaindole-BODIPY derivative to probe hydrophobic proteins as well as protein misfolding and aggregation. In organic solvents, this compound exhibits two emissive excited states efficiently interconverting. In contrast, within aqueous environments, the formation of molecular aggregates induces distinct photophysical properties. The complex photophysics of this 7-azaindole-BODIPY derivate were used as a starting point to explore its sensing applications. In presence of albumin, the monomeric form of the probe is stabilized in the hydrophobic regions of the protein, leading to a significant increase of both the fluorescence emission intensity and lifetime. A similar effect was observed when the probe interacts with protein aggregates. Notably, the fluorescence emission is less sensitive to the presence of other macromolecules such as pepsin, DNA, Ficoll 40, and coconut oil. Fluorescence lifetime imaging microscopy (FLIM) and two-photon fluorescence microscopy performed on breast cancer cells (MCF-7) and lung cancer cells (A549) incubated with this probe revealed longer fluorescence lifetimes and higher emission intensity upon oxidative stress. It is known that cellular stress leads to the accumulation and aggregation of misfolded proteins. Protein misfolding in MCF-7 cells under oxidative stress conditions was confirmed by synchrotron FTIR microspectroscopy. These results show that protein misfolding and aggregation triggered by oxidative stress can be monitored using the probe here developed.