Utilizing Malachite Green Derivatives to Diversify Fluorogen-Activating Proteins (FAPs)’ Applications

The field of cell imaging has been greatly advanced through the use of genetically encoded fluorescent probes. By genetically expressing to proteins of interest, fluorescent proteins have enabled noninvasively imaging of live cells to study protein-protein interaction, protein trafficking and protein expression and localization. In our group, we are dedicated to the development of protein reporters (FAPs) that can activate fluorescence from originally dark
molecules (fluorogens). Among these fluoromodules, FAPdL5** is a 25 kDa binder for malachite green (MG) derivatives that functions throughout living cells with thousands fold fluorescence activation and a low-picomolar dissociation constant. With rational designed MG derivatives, distinct applications have been found in single molecule imaging, physiological pH measurements
and protein detection as recombinant affinity probes. However, in previous applications, developments of functionalized MG derivatives were focused on energy-transfer pairs which a donor that signals the environmental change is usually covalently linked to MG molecule. Little
modification has been done with MG chromophore itself.
In this thesis, we explored the effects brought by introduction of different modifications on MG. First, by varying the charge of the linker, we have proposed and synthesized a series of MG derivatives with different net charge that directly alters the cell permeability. Secondly, a series of fluorinated MG derivatives were synthesized to investigate fluorination effect on spectra and affinity of MG-dL5**. Thirdly, a number of MG analogues with distinct absorption spectra were made to serve the purpose of multi-color imaging. Finally, a heavy-atom substituted MG (MG-2I)
was made, which greatly increases singlet oxygen quantum yield upon binding to dL5**. This MG-2I/dL5** complex enables protein inactivation and targeted cell killing and rapid targeted lineage ablation in living larval and adult zebrafish.