Fluorogenic NIR-probes based on 1,2,4,5-tetrazine substituted BF₂-azadipyrromethenes.

Area of Science:

• Organic Chemistry
• Materials Science
• Biochemistry

Background:

• Near-infrared (NIR) fluorophores are crucial for deep-tissue imaging due to reduced light scattering and autofluorescence.
• BF2 azadipyrromethene (NIR-AZA) dyes offer promising photophysical properties but often require functionalization for specific applications.
• Diels-Alder cycloadditions provide a bioorthogonal and efficient method for molecular activation and signal generation.

Purpose of the Study:

• To synthesize novel 1,2,4,5-tetrazine integrated NIR-AZA fluorophores.
• To investigate the impact of tetrazine substituents on fluorescence modulation and cycloaddition kinetics.
• To demonstrate the real-time imaging capabilities of these fluorophores in a microfluidic system.

Main Methods:

• Synthesis of BF2 azadipyrromethene derivatives functionalized with various 1,2,4,5-tetrazine groups (Cl, OMe, p-NO2C6H4O).
• Assessment of photophysical properties including quantum yields and fluorescence enhancement factors.
• Evaluation of Diels-Alder cycloaddition reactions with a strained alkyne substrate in different media (organic, aqueous, gels).
• Real-time imaging of the fluorogenic reaction using a continuous flow micro-reactor.

Main Results:

• Tetrazine substituents significantly influenced quantum yields, fluorescence enhancement, and cycloaddition rates.
• Efficient cycloadditions were observed in organic solvents, aqueous solutions, and gels, achieving up to 48-fold fluorescence enhancement.
• Successful real-time imaging of the "turn-on" fluorescence response was demonstrated in a microfluidic device.
• Preliminary studies suggest photoinduced electron transfer is involved in excited state quenching.

Conclusions:

• The developed tetrazine-NIR-AZA probes exhibit tunable fluorescence properties and efficient bioorthogonal activation.
• These fluorophores demonstrate significant potential for developing sensitive and real-time imaging agents.
• The findings open avenues for advanced applications in chemical biology and diagnostics.