36 publications

36 publications

Abiological Catalysis by Artificial Haem Proteins Containing Noble Metals in Place of Iron

Hartwig, J.F.

Nature 2016, 534, 534-537, 10.1038/nature17968

Enzymes that contain metal ions—that is, metalloenzymes—possess the reactivity of a transition metal centre and the potential of molecular evolution to modulate the reactivity and substrate-selectivity of the system1. By exploiting substrate promiscuity and protein engineering, the scope of reactions catalysed by native metalloenzymes has been expanded recently to include abiological transformations2,3. However, this strategy is limited by the inherent reactivity of metal centres in native metalloenzymes. To overcome this limitation, artificial metalloproteins have been created by incorporating complete, noble-metal complexes within proteins lacking native metal sites1,4,5. The interactions of the substrate with the protein in these systems are, however, distinct from those with the native protein because the metal complex occupies the substrate binding site. At the intersection of these approaches lies a third strategy, in which the native metal of a metalloenzyme is replaced with an abiological metal with reactivity different from that of the metal in a native protein6,7,8. This strategy could create artificial enzymes for abiological catalysis within the natural substrate binding site of an enzyme that can be subjected to directed evolution. Here we report the formal replacement of iron in Fe-porphyrin IX (Fe-PIX) proteins with abiological, noble metals to create enzymes that catalyse reactions not catalysed by native Fe-enzymes or other metalloenzymes9,10. In particular, we prepared modified myoglobins containing an Ir(Me) site that catalyse the functionalization of C–H bonds to form C–C bonds by carbene insertion and add carbenes to both β-substituted vinylarenes and unactivated aliphatic α-olefins. We conducted directed evolution of the Ir(Me)-myoglobin and generated mutants that form either enantiomer of the products of C–H insertion and catalyse the enantio- and diastereoselective cyclopropanation of unactivated olefins. The presented method of preparing artificial haem proteins containing abiological metal porphyrins sets the stage for the generation of artificial enzymes from innumerable combinations of PIX-protein scaffolds and unnatural metal cofactors to catalyse a wide range of abiological transformations.


Metal: Ir
Ligand type: Methyl; Porphyrin
Host protein: Myoglobin (Mb)
Anchoring strategy: Metal substitution
Optimization: Chemical & genetic
Reaction: C-H activation
Max TON: 7260
ee: 68
PDB: ---
Notes: ---

Metal: Ir
Ligand type: Methyl; Porphyrin
Host protein: Myoglobin (Mb)
Anchoring strategy: Metal substitution
Optimization: Chemical & genetic
Reaction: C-H activation
Max TON: 92
ee: 84
PDB: ---
Notes: ---

Achiral Cyclopentadienone Iron Tricarbonyl Complexes Embedded in Streptavidin: An Access to Artificial Iron Hydrogenases and Application in Asymmetric Hydrogenation

Renaud, J.-L.; Ward, T.R.

Catal. Lett. 2016, 146, 564-569, 10.1007/s10562-015-1681-6

We report on the synthesis of biotinylated (cyclopentadienone)iron tricarbonyl complexes, the in situ generation of the corresponding streptavidin conjugates and their application in asymmetric hydrogenation of imines and ketones.


Metal: Fe
Ligand type: CO; Cyclopentadienone
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical
Reaction: Hydrogenation
Max TON: 20
ee: 34
PDB: ---
Notes: ---

An Artificial Metalloenzyme with the Kinetics of Native Enzymes

Hartwig, J.F.

Science 2016, 354, 102-106, 10.1126/science.aah4427

Natural enzymes contain highly evolved active sites that lead to fast rates and high selectivities. Although artificial metalloenzymes have been developed that catalyze abiological transformations with high stereoselectivity, the activities of these artificial enzymes are much lower than those of natural enzymes. Here, we report a reconstituted artificial metalloenzyme containing an iridium porphyrin that exhibits kinetic parameters similar to those of natural enzymes. In particular, variants of the P450 enzyme CYP119 containing iridium in place of iron catalyze insertions of carbenes into C–H bonds with up to 98% enantiomeric excess, 35,000 turnovers, and 2550 hours−1 turnover frequency. This activity leads to intramolecular carbene insertions into unactivated C–H bonds and intermolecular carbene insertions into C–H bonds. These results lift the restrictions on merging chemical catalysis and biocatalysis to create highly active, productive, and selective metalloenzymes for abiological reactions.


Metal: Ir
Ligand type: Methyl; Porphyrin
Host protein: Cytochrome P450 (CYP119)
Anchoring strategy: Metal substitution
Optimization: Chemical & genetic
Reaction: C-H activation
Max TON: 582
ee: 98
PDB: ---
Notes: ---

Metal: Ir
Ligand type: Methyl; Porphyrin
Host protein: Cytochrome P450 (CYP119)
Anchoring strategy: Metal substitution
Optimization: Chemical & genetic
Reaction: C-H activation
Max TON: 35129
ee: 91
PDB: ---
Notes: ---

An Enantioselective Artificial Suzukiase Based on the Biotin–Streptavidin Technology

Ward, T.R.

Chem. Sci. 2016, 7, 673-677, 10.1039/c5sc03116h

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.


Metal: Pd
Ligand type: Allyl; Phosphine
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: 88
ee: 80
PDB: ---
Notes: ---

Metal: Pd
Ligand type: Allyl; Carbene
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: 5
ee: ---
PDB: ---
Notes: ---

An NAD(P)H-Dependent Artificial Transfer Hydrogenase for Multienzymatic Cascades

Ward, T.R.

J. Am. Chem. Soc. 2016, 138, 5781-5784, 10.1021/jacs.6b02470

Enzymes typically depend on either NAD(P)H or FADH2 as hydride source for reduction purposes. In contrast, organometallic catalysts most often rely on isopropanol or formate to generate the reactive hydride moiety. Here we show that incorporation of a Cp*Ir cofactor possessing a biotin moiety and 4,7-dihydroxy-1,10-phenanthroline into streptavidin yields an NAD(P)H-dependent artificial transfer hydrogenase (ATHase). This ATHase (0.1 mol%) catalyzes imine reduction with 1 mM NADPH (2 mol%), which can be concurrently regenerated by a glucose dehydrogenase (GDH) using only 1.2 equiv of glucose. A four-enzyme cascade consisting of the ATHase, the GDH, a monoamine oxidase, and a catalase leads to the production of enantiopure amines.


Metal: Ir
Ligand type: Cp*; Phenanthroline
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: >999
ee: >99
PDB: ---
Notes: ---

Artificial Diels–Alderase based on the Transmembrane Protein FhuA

Okuda, J.

Beilstein J. Org. Chem. 2016, 12, 1314-1321, 10.3762/bjoc.12.124

Copper(I) and copper(II) complexes were covalently linked to an engineered variant of the transmembrane protein Ferric hydroxamate uptake protein component A (FhuA ΔCVFtev). Copper(I) was incorporated using an N-heterocyclic carbene (NHC) ligand equipped with a maleimide group on the side arm at the imidazole nitrogen. Copper(II) was attached by coordination to a terpyridyl ligand. The spacer length was varied in the back of the ligand framework. These biohybrid catalysts were shown to be active in the Diels–Alder reaction of a chalcone derivative with cyclopentadiene to preferentially give the endo product.


Metal: Cu
Ligand type: Terpyridine
Host protein: FhuA
Anchoring strategy: Cystein-maleimide
Optimization: Chemical
Max TON: ---
ee: ---
PDB: ---
Notes: ---

Artificial Hydrogenases Based on Cobaloximes and Heme Oxygenase

Artero, V.

ChemPlusChem 2016, 81, 1083-1089, 10.1002/cplu.201600218

The insertion of cobaloxime catalysts in the heme‐binding pocket of heme oxygenase (HO) yields artificial hydrogenases active for H2 evolution in neutral aqueous solutions. These novel biohybrids have been purified and characterized by using UV/visible and EPR spectroscopy. These analyses revealed the presence of two distinct binding conformations, thereby providing the cobaloxime with hydrophobic and hydrophilic environments, respectively. Quantum chemical/molecular mechanical docking calculations found open and closed conformations of the binding pocket owing to mobile amino acid residues. HO‐based biohybrids incorporating a {Co(dmgH)2} (dmgH2=dimethylglyoxime) catalytic center displayed up to threefold increased turnover numbers with respect to the cobaloxime alone or to analogous sperm whale myoglobin adducts. This study thus provides a strong basis for further improvement of such biohybrids, using well‐designed modifications of the second and outer coordination spheres, through site‐directed mutagenesis of the host protein.


Metal: Co
Ligand type: Oxime
Host protein: Heme oxygenase (HO)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Reaction: H2 evolution
Max TON: 15.3
ee: ---
PDB: ---
Notes: ---

Artificial Metalloenzymes Based on the Biotin-Streptavidin Technology: Challenges and Opportunities

Review

Ward, T.R.

Acc. Chem. Res. 2016, 49, 1711-1721, 10.1021/acs.accounts.6b00235

The biotin–streptavidin technology offers an attractive means to engineer artificial metalloenzymes (ArMs). Initiated over 50 years ago by Bayer and Wilchek, the biotin–(strept)avidin techonology relies on the exquisite supramolecular affinity of either avidin or streptavidin for biotin. This versatile tool, commonly referred to as “molecular velcro”, allows nearly irreversible anchoring of biotinylated probes within a (strept)avidin host protein. Building upon a visionary publication by Whitesides from 1978, several groups have been exploiting this technology to create artificial metalloenzymes. For this purpose, a biotinylated organometallic catalyst is introduced within (strept)avidin to afford a hybrid catalyst that combines features reminiscent of both enzymes and organometallic catalysts. Importantly, ArMs can be optimized by chemogenetic means. Combining a small collection of biotinylated organometallic catalysts with streptavidin mutants allows generation of significant diversity, thus allowing optimization of the catalytic performance of ArMs. Pursuing this strategy, the following reactions have been implemented: hydrogenation, alcohol oxidation, sulfoxidation, dihydroxylation, allylic alkylation, transfer hydrogenation, Suzuki cross-coupling, C–H activation, and metathesis. In this Account, we summarize our efforts in the latter four reactions. X-ray analysis of various ArMs based on the biotin–streptavidin technology reveals the versatility and commensurability of the biotin-binding vestibule to accommodate and interact with transition states of the scrutinized organometallic transformations. In particular, streptavidin residues at positions 112 and 121 recurrently lie in close proximity to the biotinylated metal cofactor. This observation led us to develop a streamlined 24-well plate streptavidin production and screening platform to optimize the performance of ArMs. To date, most of the efforts in the field of ArMs have focused on the use of purified protein samples. This seriously limits the throughput of the optimization process. With the ultimate goal of complementing natural enzymes in the context of synthetic and chemical biology, we outline the milestones required to ultimately implement ArMs within a cellular environment. Indeed, we believe that ArMs may allow signficant expansion of the natural enzymes’ toolbox to access new-to-nature reactivities in vivo. With this ambitious goal in mind, we report on our efforts to (i) activate the biotinylated catalyst precursor upon incorporation within streptavidin, (ii) minimize the effect of the cellular environment on the ArM’s performance, and (iii) demonstrate the compatibility of ArMs with natural enzymes in cascade reactions.


Notes: ---

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

Mahy, J.-P.; Ricoux, R.

ChemBioChem 2016, 17, 433-440, 10.1002/cbic.201500445

A new artificial enzyme formed by associating NCS‐3.24 with a copper complex catalyzed the Diels–Alder cyclization of cyclopentadiene with 2‐azachalcone and led to an increase in the formation of the exo‐products. Molecular modeling proposed the preferred relative positioning of both the Trojan horse complex and the two substrates.


Metal: Cu
Ligand type: Phenanthroline
Anchoring strategy: Supramolecular
Optimization: ---
Max TON: 33
ee: ---
PDB: ---
Notes: Up to endo/exo ratio 62:38

A Structural View of Synthetic Cofactor Integration into [FeFe]-Hydrogenases

Apfel, U.-P.; Happe, T.; Kurisu, G.

Chem. Sci. 2016, 7, 959-968, 10.1039/C5SC03397G

Crystal structures of semisynthetic [FeFe]-hydrogenases with variations in the [2Fe] cluster show little structural differences despite strong effects on activity.


Metal: Fe
Ligand type: CN; CO; Dithiolate
Anchoring strategy: Dative
Optimization: Chemical
Reaction: H2 evolution
Max TON: ---
ee: ---
PDB: 4XDC
Notes: H2 evolution activity of the ArM: 2874 (mmol H2)*min-1*(mg protein)-1.

Asymmetric Catalytic Sulfoxidation by a Novel VIV8 Cluster Catalyst in the Presence of Serum Albumin: A Simple and Green Oxidation System

Bian, H.-D.; Huang, F.-P.

RSC Adv. 2016, 6, 44154-44162, 10.1039/C6RA08153C

Enantioselective oxidation of a series of alkyl aryl sulfides catalyzed by a novel VIV8 cluster is tested in an aqueous medium in the presence of serum albumin. The procedure is simple, environmentally friendly, selective, and highly reactive.


Metal: V
Anchoring strategy: Undefined
Optimization: Chemical
Reaction: Sulfoxidation
Max TON: 140
ee: 77
PDB: ---
Notes: Screening with different serum albumins.

Bovine Serum Albumin-Cobalt(II) Schiff Base Complex Hybrid: An Efficient Artificial Metalloenzyme for Enantioselective Sulfoxidation using Hydrogen Peroxide

Bian, H.-D.; Liang, H.

Dalton Trans. 2016, 45, 8061-8072, 10.1039/C5DT04507J

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.


Metal: Co
Ligand type: Amine; Phenolate
Anchoring strategy: Supramolecular
Optimization: Chemical
Reaction: Sulfoxidation
Max TON: 98
ee: 87
PDB: ---
Notes: ---

Catalysis Without a Headache: Modification of Ibuprofen for the Design of Artificial Metalloenzyme for Sulfide Oxidation

Ménage, S.

J. Mol. Catal. A: Chem. 2016, 416, 20-28, 10.1016/j.molcata.2016.02.015

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 1367 t), frequency 459 TON min−1 and selectivity (up to 69%) make this easy to use catalytic system an asset for a sustainable chemistry.


Metal: Fe
Ligand type: BPHMEN
Anchoring strategy: Supramolecular
Optimization: ---
Reaction: Sulfoxidation
Max TON: 1367
ee: ---
PDB: ---
Notes: ---

Construction of a Hybrid Biocatalyst Containing a Covalently-Linked Terpyridine Metal Complex within a Cavity of Aponitrobindin

Onoda, A.

J. Inorg. Biochem. 2016, 158, 55-61, 10.1016/j.jinorgbio.2015.12.026

A hybrid biocatalyst containing a metal terpyridine (tpy) complex within a rigid β-barrel protein nitrobindin (NB) is constructed. A tpy ligand with a maleimide group, N-[2-([2,2′:6′,2′′-terpyridin]-4′-yloxy)ethyl]maleimide (1), was covalently linked to Cys96 inside the cavity of NB to prepare a conjugate NB–1. Binding of Cu2 +, Zn2 +, or Co2 + ion to the tpy ligand in NB–1 was confirmed by UV–vis spectroscopy and ESI–TOF MS measurements. Cu2 +-bound NB–1 is found to catalyze a Diels–Alder reaction between azachalcone and cyclopentadiene in 22% yield, which is higher than that of the Cu2 +–tpy complex without the NB matrix. The results suggest that the hydrophobic cavity close to the copper active site within the NB scaffold supports the binding of the two substrates, dienophile and diene, to promote the reaction.


Metal: Cu
Ligand type: Terpyridine
Host protein: Nitrobindin (Nb)
Anchoring strategy: Cystein-maleimide
Optimization: ---
Max TON: ---
ee: ---
PDB: ---
Notes: ---

Coordination Complexes and Biomolecules: A Wise Wedding for Catalysis Upgrade

Review

Gras, E.; Hureau, C.

Coord. Chem. Rev. 2016, 308, 445-459, 10.1016/j.ccr.2015.05.011

Artificial metalloenzymes, with their high selectivity and specificity combined with a wide scope of reactivity and substrates, constitute an original approach for catalyst development. Different strategies have been proposed for their elaboration, proceeding from modification of natural enzymes using bioengineering methods to de novo protein design. Another bio-inspired methodology for the development of hybrid catalysts consists in the incorporation of coordination complexes into biomolecules, with the aim to upgrade their catalytic abilities. In these systems, the reaction performed by the naked catalyst is modulated by the well-defined structure of the host biomolecule. This conveys added value to the catalyst, such as enantioselectivity or chemoselectivity. DNA, apo-enzymes, proteins and peptides have been engaged in this approach, affording a wide diversity of reactivities and substrates. The resulting systems can then be improved by combined chemical and bioengineering optimization, allowing access to powerful catalysts. Because this approach can virtually be applied to any biomolecule or coordination complex, the elaboration of bio-based hybrid catalysts seems promising for advance in catalysis.


Notes: ---

Design and Engineering of Artificial Oxygen-Activating Metalloenzymes

Review

Lombardi, A.; Lu, Y.

Chem. Soc. Rev. 2016, 45, 5020-5054, 10.1039/C5CS00923E

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.


Notes: ---

Design Strategies for Redox Active Metalloenzymes: Applications in Hydrogen Production

Review

Ghirlanda, G.

Methods Enzymol. 2016, 389-416, 10.1016/bs.mie.2016.06.001

The last decades have seen an increased interest in finding alternative means to produce renewable fuels in order to satisfy the growing energy demands and to minimize environmental impact. Nature can serve as an inspiration for development of these methodologies, as enzymes are able to carry out a wide variety of redox processes at high efficiency, employing a wide array of earth-abundant transition metals to do so. While it is well recognized that the protein environment plays an important role in tuning the properties of the different metal centers, the structure/function relationships between amino acids and catalytic centers are not well resolved. One specific approach to study the role of proteins in both electron and proton transfer is the biomimetic design of redox active peptides, binding organometallic clusters in well-understood protein environments. Here we discuss different strategies for the design of peptides incorporating redox active FeS clusters, [FeFe]-hydrogenase organometallic mimics, and porphyrin centers into different peptide and protein environments in order to understand natural redox enzymes.


Notes: Book chapter

Directed Evolution of Artificial Metalloenzymes for In Vivo Metathesis

Panke, S.; Ward, T.R.

Nature 2016, 537, 661-665, 10.1038/nature19114

The field of biocatalysis has advanced from harnessing natural enzymes to using directed evolution to obtain new biocatalysts with tailor-made functions1. Several tools have recently been developed to expand the natural enzymatic repertoire with abiotic reactions2,3. For example, artificial metalloenzymes, which combine the versatile reaction scope of transition metals with the beneficial catalytic features of enzymes, offer an attractive means to engineer new reactions. Three complementary strategies exist3: repurposing natural metalloenzymes for abiotic transformations2,4; in silico metalloenzyme (re-)design5,6,7; and incorporation of abiotic cofactors into proteins8,9,10,11. The third strategy offers the opportunity to design a wide variety of artificial metalloenzymes for non-natural reactions. However, many metal cofactors are inhibited by cellular components and therefore require purification of the scaffold protein12,13,14,15. This limits the throughput of genetic optimization schemes applied to artificial metalloenzymes and their applicability in vivo to expand natural metabolism. Here we report the compartmentalization and in vivo evolution of an artificial metalloenzyme for olefin metathesis, which represents an archetypal organometallic reaction16,17,18,19,20,21,22 without equivalent in nature. Building on previous work6 on an artificial metallohydrolase, we exploit the periplasm of Escherichia coli as a reaction compartment for the ‘metathase’ because it offers an auspicious environment for artificial metalloenzymes, mainly owing to low concentrations of inhibitors such as glutathione, which has recently been identified as a major inhibitor15. This strategy facilitated the assembly of a functional metathase in vivo and its directed evolution with substantially increased throughput compared to conventional approaches that rely on purified protein variants. The evolved metathase compares favourably with commercial catalysts, shows activity for different metathesis substrates and can be further evolved in different directions by adjusting the workflow. Our results represent the systematic implementation and evolution of an artificial metalloenzyme that catalyses an abiotic reaction in vivo, with potential applications in, for example, non-natural metabolism.


Metal: Ru
Ligand type: Carbene
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Genetic
Reaction: Olefin metathesis
Max TON: 610
ee: ---
PDB: ---
Notes: Reaction in the periplasm

Directed Evolution of Iridium-Substituted Myoglobin Affords Versatile Artificial Metalloenzymes for Enantioselective C-C Bond-Forming Reactions

Review

Ward, T.R.

Angew. Chem. Int. Ed. 2016, 55, 14909-14911, 10.1002/anie.201607222

Upgrading myoglobin with iridium: A metal‐substitution strategy has been used to afford a repurposed myoglobin for challenging cyclopropanation and intramolecular C−H activation reactions. The performance of the iridium‐loaded myoglobin (orange sphere) was improved through directed evolution of eight active‐site residues (yellow surface).


Notes: ---

Efficient in Situ Regeneration of NADH Mimics by an Artificial Metalloenzyme

Ward, T.R.

ACS Catal. 2016, 6, 3553-3557, 10.1021/acscatal.6b00258

NADH mimics (mNADHs) have been shown to accelerate and orthogonally activate ene reductase-catalyzed reactions. However, existing regeneration methods of NAD(P)H fail for mNADHs. Catalysis with artificial metalloenzymes based on streptavidin (Sav) variants and a biotinylated iridium cofactor enable mNADH regeneration with formate. This regeneration can be coupled with ene reductase-catalyzed asymmetric reduction of α,β-unsaturated compounds, because of the protective compartmentalization of the organometallic cofactor. With 10 mol % mNAD+, a preparative scale reaction (>100 mg) gave full conversion with 98% ee, where TTNs reached 2000, with respect to the Ir cofactor under ambient atmosphere in aqueous medium.


Metal: Ir
Ligand type: Cp*; Diamine
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: >1980
ee: ---
PDB: ---
Notes: ArM works in combination with the ene reductase (ER) of the Old Yellow Enzyme family fromThermus scotuductus (TsOYE).

Evaluation of Chemical Diversity of Biotinylated Chiral 1,3-Diamines as a Catalytic Moiety in Artificial Imine Reductase

Rimoldi, I.

ChemCatChem 2016, 8, 1665-1670, 10.1002/cctc.201600116

The possibility of obtaining an efficient artificial imine reductase was investigated by introducing a chiral cofactor into artificial metalloenzymes based on biotin–streptavidin technology. In particular, a chiral biotinylated 1,3‐diamine ligand in coordination with iridium(III) complex was developed. Optimized chemogenetic studies afforded positive results in the stereoselective reduction of a cyclic imine, the salsolidine precursor, as a standard substrate with access to both enantiomers. Various factors such as pH, temperature, number of binding sites, and steric hindrance of the catalytic moiety have been proved to affect both efficiency and enantioselectivity, underlining the great flexibility of this system in comparison with the achiral system. Computational studies were also performed to explain how the metal configuration, in the proposed system, might affect the observed stereochemical outcome.


Metal: Ir
Ligand type: Amino-sulfonamide; Cp*
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: >99
ee: 83
PDB: 3PK2
Notes: ---

Genetic Optimization of Metalloenzymes: Enhancing Enzymes for Non-Natural Reactions

Review

Hyster, T.K.; Ward, T.R.

Angew. Chem. Int. Ed. 2016, 55, 7344-7357, 10.1002/anie.201508816

Artificial metalloenzymes have received increasing attention over the last decade as a possible solution to unaddressed challenges in synthetic organic chemistry. Whereas traditional transition‐metal catalysts typically only take advantage of the first coordination sphere to control reactivity and selectivity, artificial metalloenzymes can modulate both the first and second coordination spheres. This difference can manifest itself in reactivity profiles that can be truly unique to artificial metalloenzymes. This Review summarizes attempts to modulate the second coordination sphere of artificial metalloenzymes by using genetic modifications of the protein sequence. In doing so, successful attempts and creative solutions to address the challenges encountered are highlighted.


Notes: ---

Immobilization of an Artificial Imine Reductase Within Silica Nanoparticles Improves its Performance

Shahgaldian, P.; Ward, T.R.

Chem. Commun. 2016, 52, 9462-9465, 10.1039/c6cc04604e

Silica nanoparticles equipped with an artificial imine reductase display remarkable activity towards cyclic imine- and NAD+ reduction. The method, based on immobilization and protection of streptavidin on silica nanoparticles, shields the biotinylated metal cofactor against deactivation yielding over 46 000 turnovers in pure samples and 4000 turnovers in crude cellular extracts.


Metal: Ir
Ligand type: Amino-sulfonamide; Cp*
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Genetic
Max TON: 4554
ee: 89
PDB: ---
Notes: Reaction in nanoparticles

Immobilization of Two Organometallic Complexes into a Single Cage to Construct Protein-Based Microcompartment

Ueno, T.

Chem. Commun. 2016, 52, 5463-5466, 10.1039/C6CC00679E

Natural protein-based microcompartments containing multiple enzymes promote cascade reactions within cells. We use the apo-ferritin protein cage to mimic such biocompartments by immobilizing two organometallic Ir and Pd complexes into the single protein cage. Precise locations of the metals and their accumulation mechanism were studied by X-ray crystallography.


Metal: Ir
Ligand type: Amino acid; Cp*
Host protein: Apo-ferritin
Anchoring strategy: Dative
Optimization: Chemical
Reaction: Hydrogenation
Max TON: ~2
ee: 15
PDB: 5E2D
Notes: Tandem reaction (Hydrogenation and Suzuki-Miyaura coupling) to form biphenylethanol from 4-iodoacetophenone and phenylboronic acid. TON and ee are given for the tandem reaction product.

Metal: Pd
Ligand type: Allyl; Amino acid
Host protein: Apo-ferritin
Anchoring strategy: Dative
Optimization: Chemical
Max TON: ~1
ee: 15
PDB: 5E2D
Notes: Tandem reaction (Hydrogenation and Suzuki-Miyaura coupling) to form biphenylethanol from 4-iodoacetophenone and phenylboronic acid.

Library Design and Screening Protocol for Artificial Metalloenzymes Based on the Biotin-Streptavidin Technology

Ward, T.R.

Nat. Protoc. 2016, 11, 835-852, 10.1038/nprot.2016.019

Artificial metalloenzymes (ArMs) based on the incorporation of a biotinylated metal cofactor within streptavidin (Sav) combine attractive features of both homogeneous and enzymatic catalysts. To speed up their optimization, we present a streamlined protocol for the design, expression, partial purification and screening of Sav libraries. Twenty-eight positions have been subjected to mutagenesis to yield 335 Sav isoforms, which can be expressed in 24-deep-well plates using autoinduction medium. The resulting cell-free extracts (CFEs) typically contain >1 mg of soluble Sav. Two straightforward alternatives are presented, which allow the screening of ArMs using CFEs containing Sav. To produce an artificial transfer hydrogenase, Sav is coupled to a biotinylated three-legged iridium pianostool complex Cp*Ir(Biot-p-L)Cl (the cofactor). To screen Sav variants for this application, you would determine the number of free binding sites, treat them with diamide, incubate them with the cofactor and then perform the reaction with your test compound (the example used in this protocol is 1-phenyl-3,4-dihydroisoquinoline). This process takes 20 d. If you want to perform metathesis reactions, Sav is coupled to a biotinylated second-generation Grubbs-Hoveyda catalyst. In this application, it is best to first immobilize Sav on Sepharose-iminobiotin beads and then perform washing steps. Elution from the beads is achieved in an acidic reaction buffer before incubation with the cofactor. Catalysis using your test compound (in this protocol, 2-(4-(N,N-diallylsulfamoyl)phenyl)-N,N,N-trimethylethan-1-aminium iodide) is performed using the formed metalloenzyme. Screening using this approach takes 19 d.


Metal: Ir
Ligand type: Cp*; Diamine
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: 183
ee: 71
PDB: ---
Notes: Purified streptavidin (mutant K121A)

Metal: Ir
Ligand type: Cp*; Diamine
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: 42
ee: 59
PDB: ---
Notes: Cell free extract (mutant Sav K121A) treated with diamide

Metal: Ru
Ligand type: N-heterocyclic carbene
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: 66
ee: ---
PDB: ---
Notes: Purified streptavidin (mutant K121A)

Metal: Ru
Ligand type: N-heterocyclic carbene
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: 18
ee: ---
PDB: ---
Notes: Cell free extract (mutant Sav K121A immobilised on iminobiotin-sepharose beads)

Metal-Directed Design of Supramolecular Protein Assemblies

Review

Tezcan, F.A.

Methods Enzymol. 2016, 10.1016/bs.mie.2016.05.009

Owing to their central roles in cellular signaling, construction, and biochemistry, protein–protein interactions (PPIs) and protein self-assembly have become a major focus of molecular design and synthetic biology. In order to circumvent the complexity of constructing extensive noncovalent interfaces, which are typically involved in natural PPIs and protein self-assembly, we have developed two design strategies, metal-directed protein self-assembly (MDPSA) and metal-templated interface redesign (MeTIR). These strategies, inspired by both the proposed evolutionary roles of metals and their prevalence in natural PPIs, take advantage of the favorable properties of metal coordination (bonding strength, directionality, and reversibility) to guide protein self-assembly with minimal design and engineering. Using a small, monomeric protein (cytochrome cb562) as a model building block, we employed MDPSA and MeTIR to create a diverse array of functional supramolecular architectures which range from structurally tunable oligomers to metalloprotein complexes that can properly self-assemble in living cells into novel metalloenzymes. The design principles and strategies outlined herein should be readily applicable to other protein systems with the goal of creating new PPIs and protein assemblies with structures and functions not yet produced by natural evolution.


Notes: ---

Metatheases: Artificial Metalloproteins for Olefin Metathesis

Review

Okuda, J.

Org. Biomol. Chem. 2016, 14, 9174-9183, 10.1039/C6OB01475E

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.


Notes: ---

Modular Homogeneous Chromophore-Catalyst Assemblies

Review

Mulfort, K.L.

Acc. Chem. Res. 2016, 49, 835-843, 10.1021/acs.accounts.5b00539

Photosynthetic reaction center (RC) proteins convert incident solar energy to chemical energy through a network of molecular cofactors which have been evolutionarily tuned to couple efficient light-harvesting, directional electron transfer, and long-lived charge separation with secondary reaction sequences. These molecular cofactors are embedded within a complex protein environment which precisely positions each cofactor in optimal geometries along efficient electron transfer pathways with localized protein environments facilitating sequential and accumulative charge transfer. By contrast, it is difficult to approach a similar level of structural complexity in synthetic architectures for solar energy conversion. However, by using appropriate self-assembly strategies, we anticipate that molecular modules, which are independently synthesized and optimized for either light-harvesting or redox catalysis, can be organized into spatial arrangements that functionally mimic natural photosynthesis. In this Account, we describe a modular approach to new structural designs for artificial photosynthesis which is largely inspired by photosynthetic RC proteins. We focus on recent work from our lab which uses molecular modules for light-harvesting or proton reduction catalysis in different coordination geometries and different platforms, spanning from discrete supramolecular assemblies to molecule–nanoparticle hybrids to protein-based biohybrids. Molecular modules are particularly amenable to high-resolution characterization of the ground and excited state of each module using a variety of physical techniques; such spectroscopic interrogation helps our understanding of primary artificial photosynthetic mechanisms. In particular, we discuss the use of transient optical spectroscopy, EPR, and X-ray scattering techniques to elucidate dynamic structural behavior and light-induced kinetics and the impact on photocatalytic mechanism. Two different coordination geometries of supramolecular photocatalyst based on the [Ru(bpy)3]2+ (bpy = 2,2′-bipyridine) light-harvesting module with cobaloxime-based catalyst module are compared, with progress in stabilizing photoinduced charge separation identified. These same modules embedded in the small electron transfer protein ferredoxin exhibit much longer charge-separation, enabled by stepwise electron transfer through the native [2Fe-2S] cofactor. We anticipate that the use of interchangeable, molecular modules which can interact in different coordination geometries or within entirely different structural platforms will provide important fundamental insights into the effect of environment on parameters such as electron transfer and charge separation, and ultimately drive more efficient designs for artificial photosynthesis.


Notes: ---

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

Tiller, J.C.

ChemCatChem 2016, 8, 593-599, 10.1002/cctc.201501083

The 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.


Metal: Os
Ligand type: Undefined
Host protein: Laccase
Anchoring strategy: Undefined
Optimization: Chemical
Reaction: Dihydroxylation
Max TON: 842
ee: > 99
PDB: ---
Notes: ---

Oxidation Catalysis via Visible-Light Water Activation of a [Ru(bpy)3]2+ Chromophore BSA–Metallocorrole Couple

Gross, Z.; Mahy, J.-P.

Dalton Trans. 2016, 45, 706-710, 10.1039/c5dt04158a

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.


Metal: Mn
Ligand type: Corrole
Anchoring strategy: Supramolecular
Optimization: ---
Reaction: Sulfoxidation
Max TON: 21
ee: 16
PDB: ---
Notes: Water as oxygen source