Rapid Macrocycle Threading by a Fluorescent Dye-Polymer Conjugate in Water with Nanomolar Affinity

Area of Science:

• Supramolecular Chemistry
• Fluorescent Probes
• Bioconjugation

Background:

• Developing synthetic systems that mimic biological recognition is crucial for advanced diagnostics and therapeutics.
• Fluorescent probes are essential tools in biological imaging, but their sensitivity and specificity can be limited.
• Macrocyclic hosts offer unique binding pockets for guest molecules, enabling tailored molecular interactions.

Purpose of the Study:

• To develop a highly fluorescent molecular system with strong binding affinity in aqueous environments.
• To investigate the influence of polyethylene glycol (PEG) chain length on the binding kinetics and thermodynamics of a threaded dye-host complex.
• To explore the potential of this system for biological imaging applications.

Main Methods:

• Synthesis of a macrocyclic tetralactam host and a squaraine dye functionalized with polyethylene glycol (PEG) chains.
• Characterization of the binding interactions using fluorescence spectroscopy and determination of dissociation constants (Kd) in water and organic solvents.
• Measurement of bimolecular association rate constants (kon).
• Evaluation of the system's performance under cell culture conditions and imaging using fluorescence microscopy.

Main Results:

• A macrocyclic tetralactam host was successfully threaded by a fluorescent squaraine dye bearing PEG chains, exhibiting nanomolar dissociation constants in water.
• Very fast bimolecular association rates (kon ≈ 10^6–10^7 M⁻¹s⁻¹) were observed, effective under cell culture conditions.
• Significant changes in dye optical properties, including turn-on near-infrared fluorescence, were achieved, enabling cell imaging.
• Association constants in water were approximately 1000 times higher than in organic solvents, driven by favorable enthalpy.
• The rate of macrocycle threading was minimally affected by the length of the PEG chains.

Conclusions:

• The developed macrocyclic host-dye system demonstrates robust binding and enhanced fluorescence in aqueous media, suitable for biological imaging.
• The system's rapid association kinetics and tunable optical properties represent a significant advancement in fluorescent probe technology.
• This work provides a promising foundation for creating synthetic mimics of high-affinity biological recognition systems, such as streptavidin-biotin.