Non-Redox Metalloenzyme Active Site Mimetic Heterogeneous Catalysts

Recent progress in understanding the underlying mechanistic principles of biological molecular recognition events such as enzyme catalysis, antibody-antigen binding, lectin-glycoprotein interactions, and hormone-receptor interactions have triggered a rapid research development of synthetic systems with artificial molecular recognition components for medical diagnostics, drug delivery, sensors, and catalysis. The superb specificity of these biological systems derives from the precise structural and functional arrangements at the active sites provided by the supporting proteins. Our research objective is to mimic such an exquisite three-dimensional control in a non-biological system. We are particularly interested in binuclear metalloenzymes, such as urease and glucose isomerase, that can bind to weak binding substrates. As illustrated below, many crystallographic studies have shown the active sites of these enzymes comprised of hydrophilic metal binding cores surrounded by hydrophobic residues providing additional stabilization for substrate binding.

The excellent catalytic ability of these enzymes is attributed to such a high hydrophobicity contrast arrangement at the active sites. By covalently attaching hydrophilic organometallic complexes to the inner pore surfaces of some size-tunable meso-porous solids (MCM-type silicas, porous silicons, zeolites, etc.) followed by decorating the rest of pore surfaces with various hydrophobic functional groups, we can study the degree of binding enhancement between substrates and metal centers as well as the catalytic ability of these heterogeneous systems as depicted below.

"Molecular Curtains" for Protein Sensing

Many important cell adhesion events in biological systems involve small protein ligands, such as peptides and oligosaccharides, binding to their specific receptors on the cell surfaces. These binding events are usually non-redox active, multi-valent, and metal-assisted in nature. One prerequisite for developing and evaluating synthetic protein ligands as drug candidates is the availability of a universal assay to measure the strength, kinetics and metal dependence of drug-receptor binding. Thus the ability to monitor the binding in real time and obtain the kinetic details becomes crucial in designing an efficient screening assay. Unfortunately, current ELISA binding assays yield only the relative IC50 values rather than the real binding constants, making direct comparisons of binding affinities difficult. Therefore, the search for a physiologically relevant assay for both rapid inhibitor screening without tedious synthesis of complicated ELISA chimeras, and direct monitoring of binding kinetics has become important.

To design a universal fluorometric-binding sensor for studying the binding kinetics between proteins and their physiological ligands, we take advantage of the well-developed chemistry of selective modification of cyclodextrins (CDs) to covalently attach fluorescence donor and quenching groups as well as desired protein ligands to the cyclodextrin rings. By utilizing the solid state polymerization method, these functionalized CDs can be used to assemble stable inclusion complexes with linear polymeric guest molecules. The hydrophobic effect and hydrogen bonding between adjacent CD rings in these polyrotaxanes will bring the fluorophore- and quencher-labeled cyclodextrin rings together in a closely packed fashion. Since the fluorescence efficiency of energy transfer is proportional to the inverse sixth power of the separation between donor and acceptor, fluorescence of the donor CD units is quenched by the adjacent quencher CDs. When the molecular interaction between protein ligands and target proteins takes place, the protein ligand derivatized cyclodextrin rings will need to spatially separate themselves to accommodate the large protein as illustrated below.

Given that the diameters of most proteins are in the nanometer region, the fluorescence energy transfer among chromophores is hence terminated by the large distance between each CD rings. By monitoring the change of fluorescence emission intensity, the binding kinetics can be studied in real time. In addition, the multi-ligand arrangement in our system greatly resembles the multi-valent nature of the cell-adhesion events. It is our believe that the approach outlined here provides a general design principle for constructing sensitive and specific sensor systems for detection of protein toxins, antagonists, and other cell-surface protein ligands in various biologically important processes.

Conformational Change Induced Photo-Excited Electron/Energy Transfer (PET) Sensors

Acetylene based conjugated polymers (molecular wires) are known to manifest highly delocalized conjugation extending over many monomeric units. Such a reduction of the band gap was attributed to the coplanar cumulenic resonance structure at the excited states as illustrated below.

Perturbing the coplanarity between chromophores along the aromatic conjugation backbone will eliminate such cumulenic resonance structures. We are currently pursuing a methodology of general scope that will allow us to take advantage of the inorganic coordination chemistry combined with this conformational change induced perturbation of ?conjugation as the design principle of novel PET sensory system as depicted below.