Light-Up “Channel Dyes” for Haloalkane-Based Protein Labeling in Vitro and in Bacterial Cells

We describe a novel molecular strategy for engendering a strong light-up signal in fluorescence tagging of the genetically encoded HaloTag protein domain. We designed a set of haloalkane-derivatized dyes having twisted internal charge transfer (TICT) structures potentially narrow enough to partially fit into the enzyme’s haloalkane-binding channel. Testing a range of short chain lengths revealed a number of active dyes, with seven carbons yielding optimum light-up signal. The dimethylaminostilbazolium chloroheptyl dye (1d) yields a 27-fold fluorescence emission enhancement (λex = 535 nm; Em(max) = 616 nm) upon reaction with the protein. The control compound with standard 12-atom linkage shows less efficient signaling, consistent with our channel-binding hypothesis. For emission further to the red, we also prepared a chloroheptyl naphthalene-based dye; compound 2 emits at 653 nm with strong fluorescence enhancement upon reaction with the HaloTag domain. The two dyes (1d, 2) were successfully tested in wash-free imaging of protein localization in bacteria, using a HaloTag fusion of the filamenting temperature-sensitive mutant Z (FtsZ) protein in Escherichia coli (E. coli). The new dye conjugates are inexpensive and easily synthesized enzyme substrates with low background and large Stokes shifts, offering substantial benefits over known fluorescent substrates for the HaloTag enzyme.