Artificial trinuclear metallopeptidase synthesized by cross-linkage of a molecular bowl with a polystyrene derivative

A novel methodology is reported for construction of active sites of artific ial multinuclear metalloenzymes: transfer of metal-chelating sites confined in a prebuilt cage to a polymeric backbone. Artificial active sites compri sing two or three moieties of Cu(II) complex of tris(2-aminoethyl)amine (tr en) were prepared by transfer of Cu(II)tren units confined in a molecular b owl (MB) to poly(chloromethylstyrene-co-divinylbenzene) (PCD). By treatment of unreacted chloro groups of the resulting PCD with methoxide and destruc tion of the MB moieties attached to PCD with acid followed by addition of C u(II) ion to the exposed tren moieties, catalytic polymers with peptidase a ctivity were obtained. The average number (beta) of proximal Cu(II)tren moi eties in the active site of the artificial multinuclear metallopeptidase wa s determined by quantifying the Cu(II) content. Several species of the arti ficial metallopeptidases with different beta contents were prepared and exa mined for catalytic activity in hydrolysis of various cinnamoyl amide deriv atives. The PCD-based catalytic polymers did not hydrolyze a neutral amide but effectively hydrolyzed carboxyl-containing amides (N-cinnamoyl glycine, N-cinnamoyl beta-alanine, and N-cinnamoyl gamma-amino butyrate). Analysis of the kinetic data revealed that the active sites comprising three Cu(IT)t ren units were mainly responsible for the catalytic activity. When analyzed in terms of k(cat), the catalytic activity of the PCD-based artificial pep tidase was comparable to or better than the catalytic antibody with the hig hest peptidase activity reported to date. A mechanism is suggested for the effective cooperation among the three metal centers of the active site in h ydrolysis of the carboxyl-containing amides.