Artificial Metalloenzymes Research Articles

Catalysis without a headache: Modification of ibuprofen for the design of artificial metalloenzyme for sulfide oxidation

A new artificial oxidase has been developed for selective transformation of thioanisole. The catalytic activity of an iron inorganic complex, FeLibu, embedded in a transport protein NikA has been investigated in aqueous media. High efficiency (up to 1367t), frequency 459TONmin−1 and selectivity (up to 69%) make this easy to use catalytic system an asset for a sustainable chemistry.

Artificial Metalloenzymes with the Neocarzinostatin Scaffold: Toward a Biocatalyst for the Diels-Alder Reaction.

A copper(II) cofactor coupled to a testosterone anchor, copper(II)-(5-(Piperazin-1-yl)-1,10-phenanthroline)testosterone-17-hemisuccinamide (10) was synthesized and associated with a neocarzinostatin variant, NCS-3.24 (KD =3 μm), thus generating a new artificial metalloenzyme by following a "Trojan horse" strategy. Interestingly, the artificial enzyme was able to efficiently catalyze the Diels-Alder cyclization reaction of cyclopentadiene (1) with 2-azachalcone (2). In comparison with what was observed with cofactor 10 alone, the artificial enzymes favored formation of the exo products (endo/exo ratios of 84:16 and 62:38, respectively, after 12 h). Molecular modeling studies assigned the synergy between the copper complex and the testosterone (KD =13 μm) moieties in the binding of 10 to good van der Waals complementarity. Moreover, by pushing the modeling exercise to its limits, we hypothesize on the molecular grounds that are responsible for the observed selectivity.

Periplasmic Screening for Artificial Metalloenzymes.

Artificial metalloenzymes represent an attractive means of combining state-of-the-art transition metal catalysis with the benefits of natural enzymes. Despite the tremendous recent progress in this field, current efforts toward the directed evolution of these hybrid biocatalysts mainly rely on the laborious, individual purification of protein variants rendering the throughput, and hence the outcome of these campaigns feeble. We have recently developed a screening platform for the directed evolution of artificial metalloenzymes based on the streptavidin-biotin technology in the periplasm of the Gram-negative bacterium Escherichia coli. This periplasmic compartmentalization strategy comprises a number of compelling advantages, in particular with respect to artificial metalloenzymes, which lead to a drastic increase in the throughput of screening campaigns and additionally are of unique value for future in vivo applications. Therefore, we highlight here the benefits of this strategy and intend to propose a generalized guideline for the development of novel transition metal-based biocatalysts by directed evolution in order to extend the natural enzymatic repertoire.

Bovine serum albumin-cobalt(ii) Schiff base complex hybrid: an efficient artificial metalloenzyme for enantioselective sulfoxidation using hydrogen peroxide.

An artificial metalloenzyme (BSA-CoL) based on the incorporation of a cobalt(ii) Schiff base complex {CoL, H2L = 2,2'-[(1,2-ethanediyl)bis(nitrilopropylidyne)]bisphenol} with bovine serum albumin (BSA) has been synthesized and characterized. Attention is focused on the catalytic activity of this artificial metalloenzyme for enantioselective oxidation of a variety of sulfides with H2O2. The influences of parameters such as pH, temperature, and the concentration of catalyst and oxidant on thioanisole as a model are investigated. Under optimum conditions, BSA-CoL as a hybrid biocatalyst is efficient for the enantioselective oxidation of a series of sulfides, producing the corresponding sulfoxides with excellent conversion (up to 100%), chemoselectivity (up to 100%) and good enantiomeric purity (up to 87% ee) in certain cases.

Design and engineering of artificial oxygen-activating metalloenzymes.

Many efforts are being made in the design and engineering of metalloenzymes with catalytic properties fulfilling the needs of practical applications. Progress in this field has recently been accelerated by advances in computational, molecular and structural biology. This review article focuses on the recent examples of oxygen-activating metalloenzymes, developed through the strategies of de novo design, miniaturization processes and protein redesign. Considerable progress in these diverse design approaches has produced many metal-containing biocatalysts able to adopt the functions of native enzymes or even novel functions beyond those found in Nature.

An enantioselective artificial Suzukiase based on the biotin-streptavidin technology.

Introduction of a biotinylated monophosphine palladium complex within streptavidin affords an enantioselective artificial Suzukiase. Site-directed mutagenesis allowed the optimization of the activity and the enantioselectivity of this artificial metalloenzyme. A variety of atropisomeric biaryls were produced in good yields and up to 90% ee. The hybrid catalyst described herein shows comparable TOF to the previous aqueous-asymmetric Suzuki catalysts, and excellent stability under the reaction conditions to realize higher TON through longer reaction time.

Metatheases: artificial metalloproteins for olefin metathesis.

The incorporation of organometallic catalyst precursors in proteins results in so-called artificial metalloenzymes. The protein structure will control activity, selectivity and stability of the organometallic site in aqueous medium and allow non-natural reactions in biological settings. Grubbs-Hoveyda type ruthenium catalysts with an N-heterocyclic carbene (NHC) as ancillary ligand, known to be active in olefin metathesis, have recently been incorporated in various proteins. An overview of these artificial metalloproteins and their potential application in olefin metathesis is given.

Oxidation catalysis via visible-light water activation of a [Ru(bpy)3](2+) chromophore BSA-metallocorrole couple.

Light induced enantioselective oxidation of an organic molecule with water as the oxygen atom source is demonstrated in a system where chirality is induced by a protein, oxygen atom transfer by a manganese corrole, and photocatalysis by ruthenium complexes.

An internal electron reservoir enhances catalytic CO2 reduction by a semisynthetic enzyme.

The development of an artificial metalloenzyme for CO2 reduction is described. The small-molecule catalyst [Ni(II)(cyclam)](2+) has been incorporated within azurin. Selectivity for CO generation over H(+) reduction is enhanced within the protein environment, while the azurin active site metal impacts the electrochemical overpotential and photocatalytic activity. The enhanced catalysis observed for copper azurin suggests an important role for intramolecular electron transfer, analogous to native CO2 reducing enzymes.

Optimization of and Mechanistic Considerations for the Enantioselective Dihydroxylation of Styrene Catalyzed by Osmate‐Laccase‐Poly(2‐Methyloxazoline) in Organic Solvents

AbstractThe Sharpless dihydroxylation of styrene with the artificial metalloenzyme osmate‐laccase‐poly(2‐methyloxazoline) was investigated to find reaction conditions that allow this unique catalyst to reveal its full potential. After changing the co‐oxidizing agent to tert‐butyl hydroperoxide and optimizing the osmate/enzyme ratio, the turnover frequency and the turnover number could be increased by an order of magnitude, showing that the catalyst can compete with classical organometallic catalysts. Varying the metal in the active center showed that osmate is by far the most active catalytic center, but the reaction can also be realized with permanganate and iron(II) salts.

ChemInform Abstract: Schiff Base and Non‐Schiff Base Macrocyclic Ligands and Complexes Incorporating the Pyridine Moiety ‐ The First 50 Years

AbstractReview: 307 refs.

A Highly Active Biohybrid Catalyst for Olefin Metathesis in Water: Impact of a Hydrophobic Cavity in a β-Barrel Protein

A series of Grubbs–Hoveyda type catalyst precursors for olefin metathesis containing a maleimide moiety in the backbone of the NHC ligand was covalently incorporated in the cavity of the β-barrel protein nitrobindin. By using two protein mutants with different cavity sizes and choosing the suitable spacer length, an artificial metalloenzyme for olefin metathesis reactions in water in the absence of any organic cosolvents was obtained. High efficiencies reaching TON > 9000 in the ROMP of a water-soluble 7-oxanorbornene derivative and TON > 100 in ring-closing metathesis (RCM) of 4,4-bis(hydroxymethyl)-1,6-heptadiene in water under relatively mild conditions (pH 6, T = 25–40 °C) were observed.

Production and removal of superoxide anion radical by artificial metalloenzymes and redox-active metals

Generation of reactive oxygen species is useful for various medical, engineering and agricultural purposes. These include clinical modulation of immunological mechanism, enhanced degradation of organic compounds released to the environments, removal of microorganisms for the hygienic purpose, and agricultural pest control; both directly acting against pathogenic microorganisms and indirectly via stimulation of plant defense mechanism represented by systemic acquired resistance and hypersensitive response. By aiming to develop a novel classes of artificial redox-active biocatalysts involved in production and/or removal of superoxide anion radicals, recent attempts for understanding and modification of natural catalytic proteins and functional DNA sequences of mammalian and plant origins are covered in this review article.

Piano-stool d6-rhodium(III) complexes of chelating pyridine-based ligands and their papain bioconjugates for the catalysis of transfer hydrogenation of aryl ketones in aqueous medium

Two half-sandwich d6-rhodium(III) complexes of the general formula [(η5-Cp*)Rh(N^N)Cl]Cl where N^N is a phenanthroline or a bispyridine methane derivative carrying a thiol-targeting maleimide or chloroacetamide function were synthesized and characterized. Both complexes were able to catalyse the transfer hydrogenation of 2,2,2-trifluoroacetophenone in aqueous medium using formate or phosphite as hydrogen donor. Covalent anchoring of these complexes to the cysteine endoproteinase papain yielded hybrid metalloproteins with transfer hydrogenase properties. Under optimized conditions of pH, hydrogen donor concentration and catalyst load, conversion of substrate was nearly quantitative within 24h at 40°C and the (S)-enantiomer was obtained preferably albeit with a modest enantiomeric excess of 7⿿10%. Covalent docking simulations complemented the experimental findings suggesting a molecular rationale for the observed low enantioselectivity. The harmonious use of experimental and theoretical approaches represents an unprecedented starting point for driving the rational design of artificial metalloenzymes built up from papain with higher catalytic efficiency.

An artificial photosynthesis system based on CeO2 as light harvester and N-doped graphene Cu(II) complex as artificial metalloenzyme for CO2 reduction to methanol fuel

An artificial photosynthesis catalyst composed of CeO2, N-doped graphene and copper ions (CeO2–NG–Cu2+) was fabricated. The light-harvesting CeO2–NG was characterized by X-ray diffraction, transmission electron microscopy, Raman spectroscopy, and X-ray photoelectron spectroscopy. The photocatalytic reduction of CO2 was conducted in an aqueous solution of Na2SO3. Results indicated that the reduction rate of CO2 to methanol approached 507.3μmol·g−1cat.·h−1 for CeO2–NG–Cu2+ artificial photosynthesis system in 80min, whereas the reduction rate was only 5.8μmol·g−1cat.·h−1 for bare CeO2–NG without metalloenzyme. Therefore, artificial metalloenzyme played a vital role in reducing CO2 to methanol fuel.

ChemInform Abstract: Latest Developments in Metalloenzyme Design and Repurposing

AbstractReview: 89 refs.

Ring-Closing and Cross-Metathesis with Artificial Metalloenzymes Created by Covalent Active Site-Directed Hybridization of a Lipase.

A series of Grubbs-type catalysts that contain lipase-inhibiting phosphoester functionalities have been synthesized and reacted with the lipase cutinase, which leads to artificial metalloenzymes for olefin metathesis. The resulting hybrids comprise the organometallic fragment that is covalently bound to the active amino acid residue of the enzyme host in an orthogonal orientation. Differences in reactivity as well as accessibility of the active site by the functionalized inhibitor became evident through variation of the anchoring motif and substituents on the N-heterocyclic carbene ligand. Such observations led to the design of a hybrid that is active in the ring-closing metathesis and the cross-metathesis of N,N-diallyl-p-toluenesulfonamide and allylbenzene, respectively, the latter being the first example of its kind in the field of artificial metalloenzymes.

Preparation of an Immobilized Lipase‐Palladium Artificial Metalloenzyme as Catalyst in the Heck Reaction: Role of the Solid Phase

AbstractA p‐nitrophenylphosphonate palladium pincer was synthesized and selectively inserted by irreversible attachment on the catalytic serine of different commercial lipases with good to excellent yields in most cases. Among all, lipase from Candida antarctica B (CAL‐B) was the best modified enzyme. The artificial metalloenzyme CAL‐B‐palladium (Pd) catalyst was subsequently immobilized on different supports and by different orienting strategies. The catalytic properties of the immobilized hybrid catalysts were then evaluated in two sets of Heck cross‐coupling reactions under different conditions. In the first reaction between iodobenzene and ethyl acrylate, the covalent immobilized CAL‐B‐Pd catalyst resulted to be the best one exhibiting quantitative production of the Heck product at 70 °C in dimethylformamide (DMF) with 25% water and particularly in pure DMF, where the soluble Pd pincer was completely inactive. A post‐immobilization engineering of catalyst surface by its hydrophobization enhanced the activity. The selectivity properties of the best hybrid catalyst were then assessed in the asymmetric Heck cross‐coupling reaction between iodobenzene and 2,3‐dihydrofuran retrieving excellent results in terms of stereo‐ and enantioselectivity.magnified image

Improving the Catalytic Performance of an Artificial Metalloenzyme by Computational Design.

Artifical metalloenzymes combine the reactivity of small molecule catalysts with the selectivity of enzymes, and new methods are required to tune the catalytic properties of these systems for an application of interest. Structure-based computational design could help to identify amino acid mutations leading to improved catalytic activity and enantioselectivity. Here we describe the application of Rosetta Design for the genetic optimization of an artificial transfer hydrogenase (ATHase hereafter), [(η(5)-Cp*)Ir(pico)Cl] ⊂ WT hCA II (Cp* = Me5C5(-)), for the asymmetric reduction of a cyclic imine, the precursor of salsolsidine. Based on a crystal structure of the ATHase, computational design afforded four hCAII variants with protein backbone-stabilizing and hydrophobic cofactor-embedding mutations. In dansylamide-competition assays, these designs showed 46-64-fold improved affinity for the iridium pianostool complex [(η(5)-Cp*)Ir(pico)Cl]. Gratifyingly, the new designs yielded a significant improvement in both activity and enantioselectivity (from 70% ee (WT hCA II) to up to 92% ee and a 4-fold increase in total turnover number) for the production of (S)-salsolidine. Introducing additional hydrophobicity in the Cp*-moiety of the Ir-catalyst provided by adding a propyl substituent on the Cp* moiety yields the most (S)-selective (96% ee) ATHase reported to date. X-ray structural data indicate that the high enantioselectivity results from embedding the piano stool moiety within the protein, consistent with the computational model.

Supramolecular Assembly of Artificial Metalloenzymes Based on the Dimeric Protein LmrR as Promiscuous Scaffold.

Supramolecular anchoring of transition metal complexes to a protein scaffold is an attractive approach to the construction of artificial metalloenzymes since this is conveniently achieved by self-assembly. Here, we report a novel design for supramolecular artificial metalloenzymes that exploits the promiscuity of the central hydrophobic cavity of the transcription factor Lactococcal multidrug resistance Regulator (LmrR) as a generic binding site for planar coordination complexes that do not provide specific protein binding interactions. The success of this approach is manifested in the excellent enantioselectivities that are achieved in the Cu(II) catalyzed enantioselective Friedel-Crafts alkylation of indoles.