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

Abstract

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.

Fluorogenic Protein Probes with Red and Near‐Infrared Emission for Genetically Targeted Imaging**

“…The fluorogens were first coupled to the standard Halotag ligand reported in the literature to afford the Red‐Halo and NIR‐Halo compounds (Figure 1). Previous reports have shown that a shorter HaloTag ligand may increase the fluorogenicity through hydrophobic and cation‐π aromatic interactions or increased motion restriction [24, 29, 44] . We have thus synthesized an analogue of Red‐ Halo2 coupled to a shorter ligand chain with one ethylene glycol unit less: Red‐Halo2s (Figure 1).…”

“…Previous reportsh ave shownt hat as horter HaloTag ligand may increase the fluorogenicity through hydrophobic and cation-p aromatic interactions or increased motion restriction. [24,29,44] We have thus synthesized an analogue of Red-Halo2 coupled to as horter ligand chain with one ethylene glycolu nit less: Red-Halo2s ( Figure 1). We first assessed their optical properties in vitro using as tandard GST-HaloTag (GST-HT) protein ( Figure 3a nd Table S1).…”

“…Zhang and co-workers tested an analogue of the chromophore from the red fluorescent protein Kaede as af luorogenic probe targeting SNAP-tag, [28] and, for Halotag, as eries of red-emittingp yridiniums tyryl dyes called "channel dyes" have been described by Kool and co-workers. [29] In this latter report, it is assumed that the insertion of af lexible styryl dye (a typical molecular rotor) within the narrowc hannel of the HaloTag binding pocket resultsi naconstrained planar form with an increased fluorescence. The probesh ave, however, only been used in live imaging of bacteria and the use of cationic pyridiniumm ay causes electivity conflicts, since it has been reported for similar cationic dyes to accumulate in the mitochondria of eukaryotic cells.…”

“…These fluorophores display greenish fluorescence emission and very few examples exist with far‐red or near‐infrared (NIR) emissions, despite the fact that it is a highly desirable feature for in vivo imaging. Zhang and co‐workers tested an analogue of the chromophore from the red fluorescent protein Kaede as a fluorogenic probe targeting SNAP‐tag, [28] and, for Halotag, a series of red‐emitting pyridinium styryl dyes called “channel dyes” have been described by Kool and co‐workers [29] . In this latter report, it is assumed that the insertion of a flexible styryl dye (a typical molecular rotor) within the narrow channel of the HaloTag binding pocket results in a constrained planar form with an increased fluorescence.…”

The Collective Power of Genetically Encoded Protein/Peptide Tags and Bioorthogonal Chemistry in Biological Fluorescence Imaging

“…In the reported examples of fluorogenic HaloTag ligands, the dye component itself has a major non‐emissive excited state conformation which is disfavored upon ligating with the HaloTag protein to allow an emissive conformation to persist and hence to produce fluorogenicity [67] . Restricting the motion of the dye component in the binding channel of HaloTag was cited as the mechanism of fluorogenicity of the Channel Dye ( 16 in Figure 5b), [67a] while a more specific interaction with a tryptophan residue within the binding pocket has been credited with exerting a cation‐π interaction to disfavor a dark, twisted excited state and to promote a planarized emissive charge transfer state ( 17 , P9 in Figure 5b) [67c] . The Channel Dye was applied in the imaging of bacterial cells, [67a] while P9 was able to label live mammalian cells without washing steps [67c] .…”