Polymeric Artificial Metalloproteases
A R T I C L E S
of polymeric artificial enzymes, therefore, have been constructed
by using metal complexes21-28 as well as organic29-34 functional
groups.
precisely defined structure (“a catalytic module”) to a polymeric
backbone. Thus, it was attempted to synthesize new multinuclear
polymeric artificial metalloproteases by preparation of catalytic
modules containing one, two, or four metal-chelating sites
followed by attachment of the modules to a polystyrene and
addition of metal ions to the chelating sites. Previous studies
indicated that polymer backbones such as polystyrene provide
microenvironments that can enhance the intrinsic reactivity of
metal centers in the hydrolysis of peptide or phosphodiester
bonds.22,26,27 In addition, polar interactions such as electrostatic
interaction, hydrogen bonding, and dipole-dipole interaction
become stronger in more hydrophobic environments, possibly
leading to stabilization of substrate-catalyst complexes and the
transition states.
Many enzymes contain two or more metal ions in the active
site, exploiting collaboration among the metal centers in the
catalytic action. Examples of multinuclear metalloenzymes
catalyzing hydrolysis of acyl derivatives and related compounds
are methionine aminopeptidase,35 metallo-â-lactamase,36 proline
dipeptidase (prolidase),37 urease,38 and agmatinase.39 In addition,
there are a large number of multinuclear metalloenzymes that
catalyze several other types of reactions such as nucleic acid
hydrolysis, synthetic transformations, or oxidation-reduction.
An effective multinuclear artificial metalloenzyme would be
obtained if an artificial active site comprising two or more
proximal metal centers is designed. Various small molecules
with multiple metal centers have been synthesized and tested
for catalytic activity in several organic reactions including
phosphoester hydrolysis40 and asymmetric organic reactions such
as epoxidation,41 aldol condensation,42 and carbonyl reduction.43
On the other hand, construction of an artificial active site
containing two or more metal centers on a polymeric backbone
has been seldom attempted.
With an artificial enzyme obtained by attaching a prebuilt
catalytic module to a synthetic polymer, it is easier to interpret
catalytic outcome on molecular basis since the structure of the
catalytic module is known. Although several effective polymeric
artificial enzymes have been designed for protein or DNA
hydrolysis with various novel strategies to build artificial active
sites,22,26,27,30-34 detailed mechanistic analysis has not been
performed due to lack of information on exact structure of the
active sites.
In an effort to design an artificial active site comprising
multiple metal centers on the backbone of synthetic polymers,
we have reported transfer of metal-chelating sites confined in a
bowl-shaped molecule to a cross-linked polystyrene.24 The
resulting artificial active site contained three moieties of Cu-
(II) complex of tris(2-aminoethyl)amine and manifested both
catalytic activity and substrate selectivity in the hydrolysis of
small peptides. The metal centers of the artificial active site
were utilized both in substrate recognition and in catalytic
conversion: one metal center recognized the carboxylate group
of the substrate and other metal centers cleaved the peptide bond.
Little information is available, however, for the structure of the
active site obtained by using the bowl-shaped molecule. In
addition, it is not possible to synthesize a variety of artificial
multinuclear metalloenzymes by the method of transferring
catalytic elements confined in a prebuilt cage to a synthetic
polymer.
The catalytic element used in construction of the catalytic
module in the present study is the Cu(II) complex of cyclen
(Cyc). Cu(II)Cyc was chosen in view of the tight binding44 of
Cu(II) ion to Cyc at near neutral pH’s and proteolytic22 activity
of Cu(II)Cyc attached to polystyrene derivatives. As the number
of the Cu(II)Cyc units present in the catalytic module is
increased, the catalytic group density of the active site is raised.
Improvement of catalytic activity of the artificial active site
through an increase in the catalytic group density would become
a new methodology for designing artificial active sites. When
the multinuclear metal center participates in cleavage of a protein
substrate, some of the metal centers may act as the binding sites
to recognize the protein substrate and other metal centers as
catalytic groups to hydrolyze peptide bonds. If a certain
functional group of the protein substrate is recognized, then
some peptide bonds of the substrate may be selectively cleaved.
Selectivity in cleavage site is one of the important goals to
achieve at the current stage in the area of designing enzyme-
mimetic catalysts. In this article, synthesis of the multinuclear
artificial metalloproteases, kinetic data for the hydrolytic cleav-
age of myoglobin (Mb), and site-selectivity of the catalytic
action in Mb cleavage are described.
To develop a methodology applicable to designing a wide
range of multinuclear polymeric artificial metalloenzymes, we
attempted in the present study to prepare active sites by attaching
a molecular entity comprising various catalytic elements with
(29) Suh, J.; Lee, S. H.; Zoh, K. D. J. Am. Chem. Soc. 1992, 114, 7916.
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Results
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Synthesis of Catalysts. The catalytic modules containing one,
two, or four Cyc units were synthesized by attaching Cyc
(41) Finn, G.; Sharpless, K. B. J. Am. Chem. Soc. 1991, 113, 113-126,
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