Halogen Effects-Induced Bright D-π-A Fluorophore as Scaffold for NIR Fluorogenic Probes with High Contrast

Area of Science:

• Chemical Biology
• Fluorescence Imaging
• Probe Design

Background:

• Fluorogenic probes are crucial for accurate fluorescence sensing and imaging.
• Traditional phenolate-based D-π-A fluorophores (A-DOH) exhibit weak intracellular emission, leading to low contrast in live cell imaging.
• There is a need for fluorogenic probes with enhanced contrast for improved live cell imaging.

Purpose of the Study:

• To develop novel A-DOH fluorophores with bright near-infrared (NIR) fluorescence and high contrast for live cell imaging.
• To investigate the role of halogen effects in enhancing fluorescence properties.
• To demonstrate the utility of these probes for imaging specific analytes in living cells.

Main Methods:

• Synthesis of chlorinated A-DOH fluorophores (A1-OH) utilizing halogen effects.
• Investigation of fluorescence properties in aqueous solutions and living cells.
• Systematic study of the impact of halogen effects (-I and +M) on pKa and fluorescence quantum yield.
• Development of a mitochondria-targeting peroxynitrite-responsive probe (Probe-PN) based on the A1-OH scaffold.

Main Results:

• The chlorinated A1-OH fluorophore exhibited significantly enhanced fluorescence (194x in solution, 30x in cells) compared to its halogen-free counterpart.
• Halogen effects (-I and +M) were identified as key factors for reduced pKa and increased fluorescence quantum yield.
• The A1-OH scaffold allowed for the creation of various NIR fluorogenic probes with high contrast via phenol caging chemistry.
• Probe-PN demonstrated specific, high-contrast fluorescence imaging of endogenous peroxynitrite (OONO-) in macrophage mitochondria.

Conclusions:

• A general and robust approach using halogen effects was established for preparing novel A-DOH fluorophores with bright NIR fluorescence.
• The developed probes offer high contrast for live cell imaging, overcoming limitations of traditional probes.
• The A1-OH scaffold is versatile for designing functional probes for diverse biological applications, including specific analyte detection in organelles.