Electronic and Steric Optimization of Fluorogenic Probes for Biomolecular Imaging

Area of Science:

• Chemical Biology
• Cellular Imaging
• Molecular Probes

Background:

• Fluorogenic probes are essential for live-cell imaging, enabling spatiotemporal studies.
• Common probes rely on esterase hydrolysis to activate fluorescence, but are prone to spontaneous hydrolysis.
• Existing solutions involve auto-immolative linkers, which complicate synthesis and reduce atom economy.

Purpose of the Study:

• To develop a novel fluorogenic probe strategy that avoids spontaneous hydrolysis without using linkers.
• To design and synthesize probes utilizing electronic and steric interactions for improved stability and reactivity.
• To demonstrate the utility of the new probe in cellular imaging applications.

Main Methods:

• Design of linker-free fluorogenic probes incorporating specific electronic and steric features.
• Synthesis of 2',7'-dichlorofluorescein diisobutyrate.
• Evaluation of probe stability against spontaneous hydrolysis.
• Assessment of probe reactivity with esterases in vitro and in cellulo.
• Demonstration of probe performance in biomolecular imaging.

Main Results:

• A linker-free strategy was successfully implemented, enhancing probe stability.
• The probe 2',7'-dichlorofluorescein diisobutyrate exhibits optimized X···C═O n→π* interactions and acyl group size.
• The probe shows high stability against spontaneous hydrolysis.
• The probe is a reactive substrate for esterases, both in vitro and in cellulo.
• The probe generates a bright, photostable fluorophore suitable for imaging.

Conclusions:

• The developed linker-free strategy offers a significant advancement in fluorogenic probe design.
• The new probe provides a stable yet reactive platform for esterase detection in live cells.
• This approach facilitates easier synthesis and improved performance for cellular imaging applications.