Reversible and Biocompatible RedOx-Responsive Fluorescent Metallocene-based Molecular Probes – RFM

The RFM project is aimed at developing original biocompatible, versatile, tunable, and reversible redox-responsive fluorescent metallocene-based probes able to target cell membranes and/or subcellular organelles. Biocompatible reversible fluorescent probes that respond reversibly to a redox signal are an emerging but under-explored class of molecules especially for the detection of reactive oxygen and nitrogen species (ROS and RNS), which play an important role in signaling processing and in protection against pathogens. Accordingly, they would be able to distinguish transient and chronic changes in oxidative capacity in living organisms, through bio-imaging.
The probe design strategy will feature the association of an organic fluorophore of the rhodamine family, to cover a wide spectrum of emission wavelengths, and a metallocene (ferrocene, ruthenocene) in order to promote a redox-switchable Photo-induced Electron Transfer (PeT) as the key step of fluorescence control. The redox power of each metallocene will be modulated by functionalization of the cyclopentadienyl rings to target specific ROS/RNS. Further probe specific modification such as the grafting of a phospholipid chain or a chloroalkane chain will be used for membrane or for subcellular targeting via the HaloTag strategy, respectively. The reversible switching of our probes will be first investigated, both electrochemically and chemically in the presence of biologically relevant ROS/RNS and reductive species such as glutathione, not only in solution but also in the presence of artificial vesicles. Then, perspectives in bio-imaging will be evaluated in the presence of living cells.
Based on the synergistic combination of experimental (molecular electrochemistry, chemical synthesis) and theoretical investigations, the RFM project will focus on the design of the best original dyads in terms of: (i) clear fluorescent response between oxidized and reduced forms, (ii) tunability of the redox potential, (iii) selectivity, (iv) interaction/orientation in phospholipid membranes, and (v) cellular permeability/specific subcellular localization.
RFM will involve 3 partner teams (PASTEUR, LBM, i-CLeHS) in a work program divided into four main work packages: 1/ design and synthesis of metallocene-based complexes bearing rhodamine derivatives; 2/ investigations of the photo-physico-chemical properties of the various prepared probes; 3/ investigations of dyads interactions with lipid architectures at the molecular level; 4/ perspectives in bioimaging at the level of both model living cell lines and specific subcellular organelles. LBM will bring competencies in the design/synthesis of dyads and especially its long-term experience on (bio)-molecules possessing fluorescent properties. i-CLeHS will bring its strong experience in theoretical investigation, classical and quantum approaches –DFT and TD-DFT. PASTEUR will bring its expertise in molecular electrochemistry and bio-electrochemistry, as well as in biophysics and in fluorescence spectroscopy/microscopy.
The expected impact of the METAFLUOX project goes beyond the scope of this fundamental research project, addressing central issues related to theory/modeling, synthesis, analysis, and bioimaging.