Optimized Red-Absorbing Dyes for Imaging and Sensing

Abstract

Rhodamine dyes are excellent scaffolds for developing a broad range of fluorescent probes. A key property of rhodamines is their equilibrium between a colorless lactone and fluorescent zwitterion. Tuning the lactone−zwitterion equilibrium constant (K L−Z ) can optimize dye properties for specific biological applications. Here, we use known and novel organic chemistry to prepare a comprehensive collection of rhodamine dyes to elucidate the structure−activity relationships that govern K L−Z . We discovered that the auxochrome substituent strongly affects the lactone− zwitterion equilibrium, providing a roadmap for the rational design of improved rhodamine dyes. Electron-donating auxochromes, such as julolidine, work in tandem with fluorinated pendant phenyl rings to yield bright, red-shifted fluorophores for live-cell single-particle tracking (SPT) and multicolor imaging. The N-aryl auxochrome combined with fluorination yields red-shifted Forster resonance energy transfer (FRET) quencher dyes useful for creating a new semisynthetic indicator to sense cAMP using fluorescence lifetime imaging microscopy (FLIM). Together, this work expands the synthetic methods available for rhodamine synthesis, generates new reagents for advanced fluorescence imaging experiments, and describes structure−activity relationships that will guide the design of future probes.

Design of Bright Chemogenetic Reporters Based on the Combined Engineering of Fluorogenic Molecular Rotors and of the HaloTag Protein

“…[7][8][9] This approach benefits from the brightness and photostability of the rhodamine or cyanine derivatives but it relies on complex multistep synthetic pathways sometimes involving costly metal-catalyzed couplings. [10][11][12] An alternative is to use environmentally-sensitive probes, such as molecular rotors. [13,14] They are dipolar flexible structures that are not fluorescent in the free state due to non-radiative vibrational and rotational decay, but they become fluorescent when their structure is constrained in a viscous environment or when bound to biomacromolecules such as nucleic acids, proteins or membranes.…”

“…First limited to silarhodamines, the strategy has been adapted to various rhodamine scaffolds with diverse emission wavelengths and to cyanine dyes [7–9] . This approach benefits from the brightness and photostability of the rhodamine or cyanine derivatives but it relies on complex multistep synthetic pathways sometimes involving costly metal‐catalyzed couplings [10–12] . An alternative is to use environmentally‐sensitive probes, such as molecular rotors [13,14] .…”

2X-Rhodamine: A Bright and Fluorogenic Scaffold for Developing Near-Infrared Chemigenetic Indicators

“…Since the pioneering work disclosed the fluorogenic character of silicon-bridged rhodamine (SiRh), , considerable efforts have been devoted to expanding the atom-bridged rhodamine toolkit (1X-rhodamine: 1XR, Figure a). By far, only 1XRs, with bridges like XO, C­(CH 3 ) 2 , or Si­(CH 3 ) 2 , have been widely adopted due to their high K L–Z values (log K L–Z > −2), which can be finely tuned to a desired state using diverse molecular engineering techniques. ,− Examples include JF 585 /JF 635 , or MaP618/MaP700, covering the green to red spectral range. However, 1XRs with electron-withdrawing bridges like phosphine oxide (PRh) or sulfone (SO 2 Rh) have very low K L–Z (log K L–Z < −4), making them nonfluorogenic despite their long wavelengths >700 nm. − Although improved versions such as the fluorogenic JF 722 and JF 724 have become available, NIR fluorogenic dyes, especially those with emission maxima beyond 750 nm, remain largely unexplored and challenging to develop.…”

Radical reactions enabled by polyfluoroaryl fragments: photocatalysis and beyond

“…1–3 While processes relying on specific fluorine effects associated with fluoroalkyl moieties have been extensively reviewed, the ability of fluorinated aromatic groups to serve as reactivity-enabling functionality has long been neglected, and existing reviews focus on the functionalization of the polyfluoroarene core. 4–16 At the same time, polyfluoroarenes are widely used for different purposes of catalysis, 17–21 analytical chemistry, 22,23 biochemistry, 24–27 and materials science 28–31 and their applications are well beyond the C–F bond activation. This review aims to highlight the known applications of polyfluoroarenes in organic chemistry of free radicals.…”