Preparation, characterization and kinetic study of the β-cyclodextrin-based artificial metalloenzymes

Abstract

Metal ions are essential participants for the catalytic activities or structural stabilities of metalloenzymes. Metalloenzymes use the cooperative action between the substrate binding site and catalytic metal center. β-Cyclodextrin is a naturally occurring cyclic heptamer of D-(+)-glucopyranose units that are linked by $\alpha$(1→4) glucopyranose bonds. In this study, new supramolecular β-CD monomer 1 in which Cu(Ⅱ) ion binding sites are attached to the primary side of hydrophobic β-CD pockets, was prepared. β-CD dimers possessing tridentate 7- (2), pentadentate 13- (3) and 15-membered (4) pyridine diamide chelators were designed, synthesized and characterized by MALDI-MS, NMR, IR and UV-Visible spectroscopy. Fluorescence and pH-metric titration were carried out in order to ascertain their behavior as bifunctional hosts for fluorescence guests and metal ions. As expected, supramolecular β-CD dimers have high binding affinity for fluorescence guests, e.g. TNS and TPPS. Supramolecular β-CD monomer and dimers chelate Cu(Ⅱ) ion by forming amidate-Cu(Ⅱ) complexes. The $pK_{a1}$ values for the Cu(Ⅱ) promoted deprotonation of amide ligands from 1, 3 and 4 were determined to be 4.3, 6.3 and 6.3, respectively. Above pH 8.0, supramolecular ligands 3 and 4 bind fluorescent guest and Cu(Ⅱ) ion simultaneously. 1-Cu(Ⅱ), 3-Cu(Ⅱ) and 4-Cu(Ⅱ) complexes were isolated as blue or purple solids. Cu(Ⅱ) complexes catalyze the hydrolysis of p-nitrophenyl acetate, adamantate and amino acids in an enzyme-like manner; displaying Michaelis-Menten kinetics, substrate specificity and competitive inhibition. In this context, these complexes are regarded as artificial metalloenzyme. Artificial metalloenzymes hydrolyze the p-nitrophenyl esters by the nucleophilic addition of a Cu(Ⅱ)-coordinated hydroxide ion to the carbonyl carbon of substrate included in hydrophobic β-CD pocket. These results support the zinc hydroxide mechanism of carboxypeptidase A.