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

Abstract

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.