Strategic Modulation of Polarity and Viscosity Sensitivity of Bimane Molecular Rotor-Based Fluorophores for Imaging α-Synuclein
Molecular rotor-based fluorophores (RBFs) that are target-selective and sensitive to both polarity and viscosity are valuable for diverse biological applications. Here, we have designed next-generation RBFs based on the underexplored bimane fluorophore through either changing in aryl substitution or varying {pi}-linkages between the rotatable electron donors and acceptors to produce red-shifted fluorescence emissions with large Stokes shifts. RBFs exhibit a twisted intramolecular charge transfer mechanism that enables control of polarity and viscosity sensitivity, as well as target selectivity. These features enable their application in: (1) turn-on fluorescent detection of -synuclein (S) fibrils, a hallmark of Parkinsons disease (PD), including amplified fibrils from patient samples; (2) monitoring early misfolding and oligomer formation during S aggregation; and (3) selective imaging of S condensates formed by liquid-liquid phase separation (LLPS). In all three cases, we show that our probes have high levels of selectivity for S versus other aggregating proteins. These properties enable one to study the interplay of S and tau in amyloid aggregation and the mechanisms underlying neurodegenerative disorders.