Noncovalent Assembly Using a New Class of Tetralactam Macrocycles

Host-guest chemistry has diversified considerably over the past sixty years since the first report of macrocyclic crown ethers. This dissertation focuses on a class of rigid tetralactam host molecules with parallel aromatic sidewalls that can be threaded by deep-red and near-infrared squaraine and croconaine dyes with very high affinity. The term Synthavidin (synthetic avidin) technology has been coined to describe this practically useful host-guest pair. Within Synthavidin, two distinct applications have arisen resulting from the structural control of the thermodynamics and kinetics for dye threading: preassembly, which relies upon a slow rate of complex dissociation to produce long-lived fluorescent probes for biological imaging experiments, and in situ capture, which exploits high affinity and fast association kinetics for the development of diagnostic assays. The first chapter of this dissertation summarizes the thermodynamic and kinetic forces driving squaraine and croconaine threading by tetralactam macrocycles in organic and aqueous solvent, as well as previous examples of Synthavidin preassembly and in situ capture. The research presented in this dissertation focuses on optimization of new Synthavidin host-guest pairs for capture applications.

The second chapter of this dissertation describes the development of a new class of tetralactam macrocycles with parallel aromatic sidewalls and evaluation of their squaraine binding capabilities. A new tetralactam with 2,3,5,6-tetramethylphenylene sidewalls is found to thread squaraine dyes in chloroform with micromolar affinity, producing a substantial increase in deep-red fluorescence intensity. Another structurally similar tetralactam with 2,3,5,6-tetramethoxyphenylene sidewalls is found to have no squaraine affinity, as it is not suitably preorganized for threading of a squaraine guest. This preorganization is restored by restricting the conformational flexibility of these tetramethoxy groups into benzodioxole sidewalls, producing a tetralactam host with moderate affinity for squaraines, while quenching their deep-red fluorescence.

The third chapter introduces a water-soluble analogue of this 2,3,5,6- tetramethylphenylene-sidewalled tetralactam and quantifies its thermodynamic and kinetic properties for encapsulation of a variety of squaraine and croconaine dyes. This new tetralactam is found to thread squaraines and croconaines with micromolar affinity and is employed in Synthavidin preassembly alongside a targeted analogue for fluorescence imaging of cancer in vitro and in vivo. For in situ capture applications, squaraine affinity for this new macrocycle is increased to nanomolar through the supramolecular paradigm of guest back-folding.

The fourth chapter of this dissertation broadens the scope of both tetralactam hosts and fluorescent guests used in Synthavidin technology. The improved solubility of tetralactam macrocycles with 2,3,5,6-tetramethylphenylene sidewalls allows for systematic evaluation of squaraine threading by macrocycles with a variety of cationic and anionic bridgeheads. The use of a cationic tetralactam with an anionic squaraine provides a new host-guest pair with nanomolar affinity in water, leading to an additional method of modulating threading affinity. Semisquaraines and amidosquaraines are also introduced as new polar guests for tetralactam macrocycles, where their rapid threading and large increase in fluorescence upon complexation may prove very useful for the development of future capture assays.

Finally, the fifth chapter begins by describing the photophysical properties and singlet oxygen generation capability of a novel halogenated phenoxazine dye, which is found to generate a similar amount of reactive oxygen species as the commercial photosensitizer Rose Bengal and induces cell death in a clinically relevant cancer cell line. This dye is then evaluated for threading of a variety of 2,3,5,6-tetramethylphenylene tetralactam scaffolds and is found to form a complex with a tetralactam with 4-aminopiperidinium-appended bridgeheads, resulting in a characteristic color change and quenching of dye fluorescence. This fluorescence quenching is then exploited in the development of a screening assay for other possible guests for this tetralactam host, where binding of a suitable guest releases the encapsulated dye, producing a large increase in fluorescence intensity.