Design of artificial metalloenzymes

Homogeneous and enzymatic catalysis offer complementary means to generate enantiomerically pure compounds. For this reason, in a biomimetic spirit, efforts are currently under way in different groups to design artificial enzymes. Two complementary strategies are possible to incorporate active organometallic catalyst precursors into a protein environment. The first strategy utilizes covalent anchoring of the organometallic complexes into the protein environment. The second strategy relies on the use of non‐covalent incorporation of the organometallic precursor into the protein. In this review, attention is focused on the use of semisynthetic enzymes to produce efficient enantioselective hybrid catalysts for a given reaction. This article also includes our recent research results and implications in developing the biotin–avidin technology to localize the biotinylated organometallic catalyst precursor within a well‐defined protein environment. Copyright © 2005 John Wiley & Sons, Ltd.     

Bioinspired catalyst design and artificial metalloenzymes

Many bioinspired transition-metal catalysts have been developed over the recent years. In this review the progress in the design and application of ligand systems based on peptides and DNA and the development of artificial metalloenzymes are reviewed with a particular emphasis on the combination of phosphane ligands with powerful molecular recognition and shape selectivity of biomolecules. The various approaches for the assembly of these catalytic systems will be highlighted, and the possibilities that the use of the building blocks of Nature provide for catalyst optimisation strategies are discussed.     

Recent developments on creation of artificial metalloenzymes

Organic synthesis using biocatalysts has been developed over many years and is still a prominent area of research. In this context, various hybrid biocatalysts composed of a synthetic metal complex catalyst and a protein scaffold (i.e. “artificial metalloenzymes”) have been constructed. One of the most recent research areas in biocatalysts-mediated synthesis is CC bond/cleavage, the most important type of reaction in organic chemistry. Some of the artificial enzymes were applied to in-cell reactions as well as in vitro systems. The effects of the structural fluctuation in biomacromolecules on their functions have also been realized. This review article includes recent research examples of artificial metalloenzymes used to CC bond formation/cleavage. As a perspective, we also focus on how we apply protein dynamics factor for the creation of new generation artificial metalloenzymes.     

Aqueous oxidation of alcohols catalyzed by artificial metalloenzymes based on the biotin-avidin technology

Based on the incorporation of biotinylated organometallic catalyst precursors within (strept)avidin, we have developed artificial metalloenzymes for the oxidation of secondary alcohols using tert-butylhydroperoxide as oxidizing agent. In the presence of avidin as host protein, the biotinylated aminosulfonamide ruthenium piano stool complex 1 (0.4 mol%) catalyzes the oxidation of sec-phenethyl alcohol at room temperature within 90 h in over 90% yield. Gel electrophoretic analysis of the reaction mixture suggests that the host protein is not oxidatively degraded during catalysis.     

Engineered and artificial metalloenzymes for selective C–H functionalization

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Designed evolution of artificial metalloenzymes: protein catalysts made to order

Artificial metalloenzymes based on biotin-streptavidin technology, a "fusion" of chemistry and biology, illustrate how asymmetric catalysts can be improved and evolved using chemogenetic approaches.     

Merging the best of two worlds: artificial metalloenzymes for enantioselective catalysis

Artificial metalloenzymes result from combining a catalytically active organometallic moiety with a macromolecular host. The resulting hybrid catalysts combine attractive features of both homogeneous and enzymatic systems. Herein we summarize the recent progress in this emerging field and outline the challenges ahead.     

Novel artificial metalloenzymes by in vivo incorporation of metal-binding unnatural amino acids

Artificial metalloenzymes have emerged as an attractive new approach to enantioselective catalysis. Herein, we introduce a novel strategy for preparation of artificial metalloenzymes utilizing amber stop codon suppression methodology for the in vivo incorporation of metal-binding unnatural amino acids. The resulting artificial metalloenzymes were applied in catalytic asymmetric Friedel–Crafts alkylation reactions and up to 83% ee for the product was achieved.     

Chemogenetic protein engineering: an efficient tool for the optimization of artificial metalloenzymes

Artificial metalloenzymes, based on the incorporation of a catalytically active organometallic moiety within a host protein, lie at the interface between organometallic and enzymatic catalysis. In terms of activity, reaction repertoire, substrate range and operating conditions, they take advantage of the versatility of the organometallic chemistry. In contrast, the enantioselectivity is determined by the biomolecular scaffold, which provides a well defined second coordination sphere to the organometallic moiety, reminiscent of enzymes. The attractive feature of such systems is their optimization potential, which combines chemical and genetic methods (i.e. chemogenetic) to screen diversity space. This feature article describes the implementation of such an optimization protocol for artificial transfer hydrogenases, for which we have the most detailed understanding.     

Rapid determination of volatile organic compounds in environmentally hazardous wastewaters using solid phase microextraction

Solid phase micro-extraction (SPME) was applied to the determination of volatile organic compounds (VOC) in wastewater discharges. Environmentally significant samples, typical of those subject to regulatory control, were examined and included discharges from pharmaceutical, petrochemical and municipal sewerage treatment plants. Analysis was performed using gas chromatography – mass spectrometry (GC-MS) following sampling using headspace or immersion SPME. Fused silica fibres, coated with either poly(dimethylsiloxane) or poly(acrylate), were examined to determine VOC which included chloroform, saturated carboxylic acids, alkylbenzenes, phenol, benzonitrile and benzofuran. Detection limits varied from 10 to 170 ng/ml and satisfactory relative standard deviations (%RSD < 10) were obtained. For most samples, headspace sampling was preferred to immersion. SPME was found to be a useful technique for the rapid screening of wastewaters for VOC.     

Incorporation of biotinylated manganese-salen complexes into streptavidin: New artificial metalloenzymes for enantioselective sulfoxidation

Incorporation of achiral biotinylated manganese-salen complexes into streptavidin yields artificial metalloenzymes for aqueous sulfoxidation using hydrogen peroxide. Four biotinylated salen ligands were synthesized and their manganese complexes were tested in combination with several streptavidin mutants, yielding moderate conversions (up to 56%) and low enantioselectivities (maximum of 13% ee) for the sulfoxidation of thioanisole.     

Progress toward artificial metalloenzymes: New ligands for transition metal ions and neutral molecules

New nickel catalysts have been developed for the oxidation of alkenes to epoxides, alcohols, aldehydes and ketones. Mechanistic studies indicate that the oxidation reactions are very sensitive to the nature of the catalyst; only certain ligands including salen and the macrocycles cyclam and dioxocyclam render Ni(II) effective as a catalyst. A Ni(III) or Ni(IV)-oxo species has been postulated as the catalytically active oxidant which leads to oxygen atom transfer to alkenes in a stepwise process. Both iodosylbenzene and hypochlorite have been used as terminal oxidants; both systems give high yields of epoxidation of alkenes and varying amounts of C=C bond cleavage products. In order to reach an ultimate goal of hydrocarbon oxidation within a molecular recognition system, new molecular receptors for organic substrates have been investigated. The receptors are constructed from two subunits of cholic acid and display amphophilic character — a hydrophobic exterior and a hydrophilic interior. Conformational properties in the presence of polar guests in CDCl₃ are described.     

Design of artificial metalloenzymes using non-covalent insertion of a metal complex into a protein scaffold

Construction of artificial metalloenzymes is one of the most attractive targets in the field of inorganic and catalytic chemistry, since they show remarkable chemoselectivity and reactivity in aqueous media. For the purpose, covalent modification of protein and cofactors have usually been utilized to attach a metal complex(es) to a protein scaffold. This article focuses on non-covalent insertion of metal complexes into protein environments. The discussion includes the screening of stable metal complex/protein composites, crystal structures, molecular design for regulating enantioselectivity of the target catalytic reactions. Our recent results show that the non-covalent conjugation will provide us a new way in semi-synthesis of artificial metalloenzymes.     

Oxidations by copper metalloenzymes and some biomimetic approaches

This paper illustrates the various aspects of the reactivity of the Cu(II)–Cu(I) system in biological systems, with one example of an enzymatic reaction in which Cu(II) alone is oxidizing enough to carry out the reaction (superoxide dismutase), one example in which a Cu(II)-bound peroxo intermediate is the active species (tyrosinase) and the examples of galactose oxidase and copper amine oxidases in which Cu(II) is associated with a redox active organic cofactor. In some cases, we will show some illustrations of biomimetic approaches developed in our laboratories, aimed at a better understanding of reaction mechanisms and at an original design of new catalysts with potential applications in synthetic chemistry. Some comments are given concerning the respective features of copper and iron.     

Modeling aspects of mechanisms for reactions catalyzed by metalloenzymes

Different models to treat metal‐catalyzed enzyme reactions are investigated. The test case chosen is the recently suggested full catalytic cycle of manganese catalase including eight different steps. This cycle contains O? O and O? H activations, as well as electron transfer steps and redox active reactions, and is therefore believed to be representative of many similar systems. Questions concerning modeling of ligands and the accuracy of the computational model are studied. Imidazole modeling of histidines are compared to ammonia modeling, and formate modeling compared to acetate modeling of glutamates. The basis set size required for the geometry optimization and for the final energy evaluation is also investigated. The adequacy of the model is judged in relation to the inherent accuracy achievable with the hybrid DFT method B3LYP. © 2001 John Wiley & Sons, Inc. J Comput Chem 22: 1634–1645, 2001     

Quantitative analysis of immobilized metalloenzymes by atomic absorption spectroscopy

A new, sensitive assay for the quantitative determination of immobilized metal containing enzymes has been developed using atomic absorption spectroscopy (AAS). In contrast with conventionally used indirect methods the described quantitative AAS assay for metalloenzymes allows more exact analyses, because the carrier material with the enzyme is investigated directly. As an example, the validity and reliability of the method was examined by fixing the iron-containing enzyme catalase on cotton fabrics using different immobilization techniques. Sample preparation was carried out by dissolving the loaded fabrics in sulfuric acid before oxidising the residues with hydrogen peroxide. The iron concentrations were determined by flame atomic absorption spectrometry after calibration of the spectrometer with solutions of the free enzyme at different concentrations.     

Rapid CE‐UV binding tests of environmentally hazardous compounds with polymer‐modified magnetic nanoparticles

Hazardous compounds and bacteria in water have an adverse impact on human health and environmental ecology. Polydopamine (or polypyrrole)‐coated magnetic nanoparticles and polymethacrylic acid‐co‐ethylene glycol dimethacrylate submicron particles were investigated for their fast binding kinetics with bisphenol A, proflavine, naphthalene acetic acid, and Escherichia coli. A new method was developed for the rapid determination of % binding by sequential injection of particles first and compounds (or E. coli) next into a fused‐silica capillary for overlap binding during electrophoretic migration. Only nanolitre volumes of compounds and particles were sufficient to complete a rapid binding test. After heterogeneous binding, separation of the compounds from the particles was afforded by capillary electrophoresis. % binding was influenced by applied voltage but not current flow. In‐capillary coating of particles affected the % binding of compounds.