Bis(trifluoromethyl)carborhodamines: Highly Fluorogenic, Far-Red to Near-Infrared Dyes for Live Cell Fluorescence Microscopy, Activity-Based Sensing, and Single-Molecule Microscopy

Synthetic fluorophores built on a classic rhodamine scaffold are essential for modern microscopy. An attractive feature of synthetic fluorophores is their potential to access long-wavelength excitation and emission profiles (>650 nm) that are difficult to achieve through genetically encoded methods like fluorescent proteins. Here, we present a new strategy to achieve excitation and emission above 650 nm: bis(trifluoromethyl)carborhodamine dyes, or BF dyes. In BF dyes, the geminal methyl groups of carborhodamines are replaced with trifluoromethyl (CF3) groups. This accomplishes two things. First, CF3 groups substantially red shift in the optical profile by over 90 nm compared to classic, oxygen-bridged rhodamine dyes, resulting in a dye framework with excitation and emission profiles >650 nm and high brightness (extinction coefficient >140,000 M-1 cm-1 and fluorescent quantum yield of 33%). Second, CF3 groups render BF dyes fluorogenic, by shifting the position of the open-closed equilibrium of the colorless lactone and colored zwitterion form, resulting in up to a 30-fold improvement in fluorogenicity compared to silicon-bridged rhodamines. In this paper, we present the design and computational analysis of BF dyes; synthetic studies to access over a dozen new BF dyes through a unique, late-stage functionalization strategy; spectra characterization; and applications in advanced fluorescence microscopy including no-wash intracellular labeling, functional imaging with chemigenetic indicators, and single molecule tracking in living cells. Together, this report shows that bis(trifluoromethyl)carborhodamine dyes provide a complementary approach to achieving long-wavelength, fluorogenic dyes for live cell microscopy that do not rely on dimethyl silicon rhodamines.