82 publications

82 publications

8-Amino-5,6,7,8-tetrahydroquinoline in Iridium(III) Biotinylated Cp* Complex as Artificial Imine Reductase

Rimoldi, I.

New J. Chem. 2018, 42, 18773-18776, 10.1039/C8NJ04558E

The imine reductase formed by the (R)-CAMPY ligand bound to the S112M Sav mutant showed an 83% ee in the asymmetric transfer hydrogenation of 6,7-dimethoxy-1-methyl-3,4-dihydroisoquinoline.


Metal: Ir
Ligand type: Cp*; Diamine
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: 32
ee: 83
PDB: ---
Notes: ---

Metal: Ir
Ligand type: Cp*; Diamine
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: 99
ee: 13
PDB: ---
Notes: ---

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: ---

Active Site Topology of Artificial Peroxidase-like Hemoproteins Based on Antibodies Constructed from a Specifically Designed Ortho-carboxy-substituted Tetraarylporphyrin

Mahy, J.-P.

Eur. J. Biochem. 1998, 257, 121-130, 10.1046/j.1432-1327.1998.2570121.x

The topology of the binding site has been studied for two monoclonal antibodies 13G10 and 14H7, elicited against iron(III)‐α,α,α,β‐meso‐tetrakis(ortho‐carboxyphenyl)porphyrin {α,α,α,β‐Fe[(o‐COOHPh)4‐porphyrin]}, and which exhibit in the presence of this α,α,α,β‐Fe[(o‐COOHPh)4‐porphyrin] cofactor a peroxidase activity. A comparison of the dissociation constants of the complexes of 13G10 and 14H7 with various tetra‐aryl‐substituted porphyrin has shown that : (a) the central iron(III) atom of α,α,α,β‐Fe[(o‐COOHPh)4‐porphyrin] is not recognized by either of the two antibodies; and (b) the ortho‐carboxylate substituents of the meso‐phenyl rings of α,α,α,β‐Fe[(o‐COOHPh)4‐porphyrin] are essential for the recognition of the porphyrin by 13G10 and 14H7. Measurement of the dissociation constants for the complexes of 13G10 and 14H7 with the four atropoisomers of (o‐COOHPh)4‐porphyrinH2 as well as mono‐ and di‐ortho‐carboxyphenyl‐substituted porphyrins suggests that the three carboxylates in the α, α, β position are recognized by both 13G10 and 14H7 with the two in the α, β positions more strongly bound to the antibody protein. Accordingly, the topology of the active site of 13G10 and 14H7 has roughly two‐thirds of the α,α,α,β‐Fe[(o‐COOHPh)4‐porphyrin] cofactor inserted into the binding site of the antibodies, with one of the aryl ring remaining outside. Three of the carboxylates are bound to the protein but no amino acid residue acts as an axial ligand to the iron atom. Chemical modification of lysine, histidine, tryptophan and arginine residues has shown that only modification of arginine residues causes a decrease in both the binding of α,α,α,β‐Fe[(o‐COOHPh)4‐porphyrin] and the peroxidase activity of both antibodies. Consequently, at least one of the carboxylates of the hapten is bound to an arginine residue and no amino acids such as lysine, histidine or tryptophan participate in the catalysis of the heterolytic cleavage of the O‐O bond of H2O2. In addition, the amino acid sequence of both antibodies not only reveals the presence of arginine residues, which could be those involved in the binding of the carboxylates of the hapten, but also the presence of several amino acids in the complementary determining regions which could bind other carboxylates through a network of H bonds.


Metal: Fe
Ligand type: ---
Host protein: Antibody 13G10 / 14H7
Anchoring strategy: Antibody
Optimization: Chemical & genetic
Reaction: Peroxidation
Max TON: ---
ee: ---
PDB: ---
Notes: ---

A De Novo Designed Metalloenzyme for the Hydration of CO2

Pecoraro, V.L.

Angew. Chem. Int. Ed. 2014, 53, 7900-7903, 10.1002/anie.201404925

Protein design will ultimately allow for the creation of artificial enzymes with novel functions and unprecedented stability. To test our current mastery of nature’s approach to catalysis, a ZnII metalloenzyme was prepared using de novo design. α3DH3 folds into a stable single‐stranded three‐helix bundle and binds ZnII with high affinity using His3O coordination. The resulting metalloenzyme catalyzes the hydration of CO2 better than any small molecule model of carbonic anhydrase and with an efficiency within 1400‐fold of the fastest carbonic anhydrase isoform, CAII, and 11‐fold of CAIII.


Metal: Zn
Ligand type: Amino acid
Host protein: α3D peptide
Anchoring strategy: Dative
Optimization: Chemical & genetic
Max TON: ---
ee: ---
PDB: ---
Notes: kcat/KM ≈ 3.8*104 M-1*s-1

A Designed Functional Metalloenzyme that Reduces O2 to H2O with Over One Thousand Turnovers

Lu, Y.

Angew. Chem. Int. Ed. 2012, 51, 5589-5592, 10.1002/anie.201201981

Rational design of functional enzymes with a high number of turnovers is a challenge, especially those with a complex active site, such as respiratory oxidases. Introducing two His and one Tyr residues into myoglobin resulted in enzymes that reduce O2 to H2O with more than 1000 turnovers (red line, see scheme) and minimal release of reactive oxygen species. The positioning of the Tyr residue is critical for activity.


Metal: Cu
Ligand type: Amino acid
Host protein: Myoglobin (Mb)
Anchoring strategy: Dative
Optimization: Chemical & genetic
Max TON: 1056
ee: ---
PDB: 4FWX
Notes: Sperm whale myoglobin

A Designed Heme-[4Fe-4S] Metalloenzyme Catalyzes Sulfite Reduction like the Native Enzyme

Lu, Y.

Science 2018, 361, 1098-1101, 10.1126/science.aat8474

Multielectron redox reactions often require multicofactor metalloenzymes to facilitate coupled electron and proton movement, but it is challenging to design artificial enzymes to catalyze these important reactions, owing to their structural and functional complexity. We report a designed heteronuclear heme-[4Fe-4S] cofactor in cytochrome c peroxidase as a structural and functional model of the enzyme sulfite reductase. The initial model exhibits spectroscopic and ligand-binding properties of the native enzyme, and sulfite reduction activity was improved—through rational tuning of the secondary sphere interactions around the [4Fe-4S] and the substrate-binding sites—to be close to that of the native enzyme. By offering insight into the requirements for a demanding six-electron, seven-proton reaction that has so far eluded synthetic catalysts, this study provides strategies for designing highly functional multicofactor artificial enzymes.


Metal: Fe
Host protein: Cytochrome c peroxidase
Anchoring strategy: Dative
Optimization: Chemical & genetic
Reaction: Sulfite reduction
Max TON: ---
ee: ---
PDB: ---
Notes: Designed heteronuclear heme-[4Fe-4S] cofactor in cytochrome c peroxidase

Albumin-Conjugated Corrole Metal Complexes: Extremely Simple Yet Very Efficient Biomimetic Oxidation Systems

Gross, Z.

J. Am. Chem. Soc. 2005, 127, 2883-2887, 10.1021/ja045372c

An extremely simple biomimetic oxidation system, consisting of mixing metal complexes of amphiphilic corroles with serum albumins, utilizes hydrogen peroxide for asymmetric sulfoxidation in up to 74% ee. The albumin-conjugated manganese corroles also display catalase-like activity, and mechanistic evidence points toward oxidant-coordinated manganese(III) as the prime reaction intermediate.


Metal: Mn
Ligand type: Corrole
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Reaction: Sulfoxidation
Max TON: 8
ee: 74
PDB: ---
Notes: ---

Metal: Mn
Ligand type: Corrole
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Reaction: Sulfoxidation
Max TON: 42
ee: 52
PDB: ---
Notes: ---

An Artificial Heme Enzyme for Cyclopropanation Reactions

Roelfes, G.

Angew. Chem. Int. Ed. 2018, 57, 7785-7789, 10.1002/anie.201802946

An artificial heme enzyme was created through self‐assembly from hemin and the lactococcal multidrug resistance regulator (LmrR). The crystal structure shows the heme bound inside the hydrophobic pore of the protein, where it appears inaccessible for substrates. However, good catalytic activity and moderate enantioselectivity was observed in an abiological cyclopropanation reaction. We propose that the dynamic nature of the structure of the LmrR protein is key to the observed activity. This was supported by molecular dynamics simulations, which showed transient formation of opened conformations that allow the binding of substrates and the formation of pre‐catalytic structures.


Metal: Fe
Ligand type: Protoporphyrin IX
Host protein: LmrR
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Reaction: Cyclopropanation
Max TON: 449
ee: 51
PDB: 6FUU
Notes: ---

An Artificial Metalloenzyme for Carbene Transfer Based on a Biotinylated Dirhodium Anchored Within Streptavidin

Ward, T.R.

Cat. Sci. Technol. 2018, 8, 2294-2298, 10.1039/C8CY00646F

We report an artificial carbenoid transferase which combines a biotinylated dirhodium moiety within streptavidin scaffold.


Metal: Rh
Ligand type: Carboxylate
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Reaction: Cyclopropanation
Max TON: ~60
ee: ---
PDB: ---
Notes: Cyclopropanation reaction was also performed in the E. coli periplasm.

Metal: Rh
Ligand type: Carboxylate
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Reaction: C-H insertion
Max TON: ~60
ee: ---
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: ---

A Noncanonical Proximal Heme Ligand Affords an Efficient Peroxidase in a Globin Fold

Green, A.P.; Hilvert, D.

J. Am. Chem. Soc. 2018, 140, 1535-1543, 10.1021/jacs.7b12621

Expanding the range of genetically encoded metal coordination environments accessible within tunable protein scaffolds presents excellent opportunities for the creation of metalloenzymes with augmented properties and novel activities. Here, we demonstrate that installation of a noncanonical Nδ-methyl histidine (NMH) as the proximal heme ligand in the oxygen binding protein myoglobin (Mb) leads to substantial increases in heme redox potential and promiscuous peroxidase activity. Structural characterization of this catalytically modified myoglobin variant (Mb NMH) revealed significant changes in the proximal pocket, including alterations to hydrogen-bonding interactions involving the prosthetic porphyrin cofactor. Further optimization of Mb NMH via a combination of rational modification and several rounds of laboratory evolution afforded efficient peroxidase biocatalysts within a globin fold, with activities comparable to those displayed by nature’s peroxidases.


Metal: Fe
Host protein: Myoglobin (Mb)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Reaction: Oxidation
Max TON: ~1650
ee: ---
PDB: 5OJ9
Notes: Oxidation of amplex red

Aqueous Oxidation of Alcohols Catalyzed by Artificial Metalloenzymes Based on the Biotin–Avidin Technology

Ward, T.R.

J. Organomet. Chem. 2005, 690, 4488-4491, 10.1016/j.jorganchem.2005.02.001

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.


Metal: Ru
Ligand type: Amino-sulfonamide; Benzene
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Reaction: Alcohol oxidation
Max TON: 200
ee: ---
PDB: ---
Notes: ---

Metal: Ru
Ligand type: Amino-sulfonamide; Benzene
Host protein: Avidin (Av)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Reaction: Alcohol oxidation
Max TON: 230
ee: ---
PDB: ---
Notes: ---

Metal: Ru
Ligand type: Bipyridine; C6Me6
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Reaction: Alcohol oxidation
Max TON: 173
ee: ---
PDB: ---
Notes: ---

Metal: Rh
Ligand type: Amino-sulfonamide; Cp*
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Reaction: Alcohol oxidation
Max TON: 7.5
ee: ---
PDB: ---
Notes: ---

Metal: Ir
Ligand type: Bipyridine; Cp*
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Reaction: Alcohol oxidation
Max TON: 30
ee: ---
PDB: ---
Notes: ---

Artificial Copper Enzymes for Asymmetric Diels–AlderReactions

Kamer, P.C.J.; Laan, W.

ChemCatChem 2013, 5, 1184-1191, 10.1002/cctc.201200671

The development of artificial copper enzymes from sterol carrier protein type 2 like domain (SCP‐2L) for the use in asymmetric catalysis was explored. For this purpose, proteins were modified with various nitrogen donor ligands. Maleimide‐containing ligands were found most suitable for selective cysteine bio‐conjugation. Fluorescence spectroscopy was used to confirm copper binding to an introduced phenanthroline ligand, which was introduced in two unique cysteine containing SCP‐2L mutants. Copper adducts of several modified SCP‐2L templates were applied in asymmetric Diels–Alder reactions. A clear influence of both the protein environment and the introduced ligand was found in the asymmetric Diels–Alder reaction between azachalcone and cyclopentadiene. A promising enantioselectivity of 25 % ee was obtained by using SCP‐2L V83C modified with phenanthroline–maleimide ligand. Good endo selectivity was observed for SCP‐2L modified with the dipicolylamine‐based nitrogen donor ligand. These artificial metalloenzymes provide a suitable starting point for the implementation of various available techniques to optimise the performance of this system.


Metal: Cu
Anchoring strategy: Covalent
Optimization: Chemical & genetic
Max TON: 9.6
ee: 25
PDB: 1IKT
Notes: ---

Artificial Heme Enzymes for the Construction of Gold-Based Biomaterials

Lombardi, A.; Nastri, F.

Int. J. Mol. Sci. 2018, 19, 2896, 10.3390/ijms19102896

Many efforts are continuously devoted to the construction of hybrid biomaterials for specific applications, by immobilizing enzymes on different types of surfaces and/or nanomaterials. In addition, advances in computational, molecular and structural biology have led to a variety of strategies for designing and engineering artificial enzymes with defined catalytic properties. Here, we report the conjugation of an artificial heme enzyme (MIMO) with lipoic acid (LA) as a building block for the development of gold-based biomaterials. We show that the artificial MIMO@LA can be successfully conjugated to gold nanoparticles or immobilized onto gold electrode surfaces, displaying quasi-reversible redox properties and peroxidase activity. The results of this work open interesting perspectives toward the development of new totally-synthetic catalytic biomaterials for application in biotechnology and biomedicine, expanding the range of the biomolecular component aside from traditional native enzymes.


Metal: Fe
Ligand type: Amino acid; Porphyrin
Anchoring strategy: Covalent
Optimization: Chemical & genetic
Reaction: Oxidation
Max TON: ---
ee: ---
PDB: ---
Notes: Immobilization of the ArM on gold surfaces via a lipoic acid anchor.

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 as Catalysts for Oxidative Lignin Degradation

Jarvis, A.G.

ACS Sustainable Chem. Eng. 2018, 6, 15100-15107, 10.1021/acssuschemeng.8b03568

We report novel artificial metalloenzymes (ArMs), containing tris(pyridylmethyl)amine (TPA), for the atom economic oxidation of lignin β-O-4 model compounds, using hydrogen peroxide. The protein scaffold alters the selectivity of the reaction from a low yielding cleavage reaction when using the parent Fe-tpa complex to a high yielding benzylic alcohol oxidation when using the complex incorporated into a protein scaffold, SCP-2L A100C. Engineering the protein scaffold to incorporate glutamic acid was found to improve the ArM activity, showing that rational design of the protein environment using metal binding amino acids can be a first step toward improving the overall activity of an artificial metalloenzyme.


Metal: Fe
Anchoring strategy: Cystein-maleimide
Optimization: Chemical & genetic
Reaction: Lignin oxidation
Max TON: 20
ee: ---
PDB: ---
Notes: Reaction performed with a lignin model compound and hydrogen peroxide as oxidizing agent

Artificial Metalloenzymes Based on Biotin-Avidin Technology for the Enantioselective Reduction of Ketones by Transfer Hydrogenation

Ward, T.R.

Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 4683-4687, 10.1073/pnas.0409684102

Most physiological and biotechnological processes rely on molecular recognition between chiral (handed) molecules. Manmade homogeneous catalysts and enzymes offer complementary means for producing enantiopure (single-handed) compounds. As the subtle details that govern chiral discrimination are difficult to predict, improving the performance of such catalysts often relies on trial-and-error procedures. Homogeneous catalysts are optimized by chemical modification of the chiral environment around the metal center. Enzymes can be improved by modification of gene encoding the protein. Incorporation of a biotinylated organometallic catalyst into a host protein (avidin or streptavidin) affords versatile artificial metalloenzymes for the reduction of ketones by transfer hydrogenation. The boric acid·formate mixture was identified as a hydrogen source compatible with these artificial metalloenzymes. A combined chemo-genetic procedure allows us to optimize the activity and selectivity of these hybrid catalysts: up to 94% (R) enantiomeric excess for the reduction of p-methylacetophenone. These artificial metalloenzymes display features reminiscent of both homogeneous catalysts and enzymes.


Metal: Ru
Ligand type: Amino-sulfonamide; P-cymene
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: 92
ee: 94
PDB: ---
Notes: ---

Metal: Ru
Ligand type: Amino-sulfonamide; Benzene
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: 30
ee: 63
PDB: ---
Notes: ---

Artificial Metalloenzymes for Asymmetric Allylic Alkylation on the Basis of the Biotin–Avidin Technology

Ward, T.R.

Angew. Chem. Int. Ed. 2008, 47, 701-705, 10.1002/anie.200703159

Palladium in the active site: The incorporation of a biotinylated palladium diphosphine within streptavidin yielded an artificial metalloenzyme for the title reaction (see scheme). Chemogenetic optimization of the catalyst by the introduction of a spacer (red star) between biotin (green triangle) and palladium and saturation mutagenesis at position S112X afforded both R‐ and S‐selective artificial asymmetric allylic alkylases.


Metal: Pd
Ligand type: Phosphine
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Reaction: Allylic alkylation
Max TON: 10
ee: 93
PDB: ---
Notes: ---

Artificial Metalloenzymes for Enantioselective Catalysis Based on Biotin-Avidin

Ward, T.R.

J. Am. Chem. Soc. 2003, 125, 9030-9031, 10.1021/ja035545i

Homogeneous and enzymatic catalysis offer complementary means to generate enantiomerically pure compounds. Incorporation of achiral biotinylated rhodium−diphosphine complexes into (strept)avidin yields artificial metalloenzymes for the hydrogenation of N-protected dehydroamino acids. A chemogenetic optimization procedure allows one to produce (R)-acetamidoalanine with 96% enantioselectivity. These hybrid catalysts display features reminiscent both of enzymatic and of homogeneous systems.


Metal: Rh
Ligand type: Phosphine
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Reaction: Hydrogenation
Max TON: ---
ee: 96
PDB: ---
Notes: ---

Artificial Metalloenzymes: (Strept)avidin as Host for Enantioselective Hydrogenation by Achiral Biotinylated Rhodium-Diphosphine Complexes

Ward, T.R.

J. Am. Chem. Soc. 2004, 126, 14411-14418, 10.1021/ja0476718

We report on the generation of artificial metalloenzymes based on the noncovalent incorporation of biotinylated rhodium−diphosphine complexes in (strept)avidin as host proteins. A chemogenetic optimization procedure allows one to optimize the enantioselectivity for the reduction of acetamidoacrylic acid (up to 96% ee (R) in streptavidin S112G and up to 80% ee (S) in WT avidin). The association constant between a prototypical cationic biotinylated rhodium−diphosphine catalyst precursor and the host proteins was determined at neutral pH:  log Ka = 7.7 for avidin (pI = 10.4) and log Ka = 7.1 for streptavidin (pI = 6.4). It is shown that the optimal operating conditions for the enantioselective reduction are 5 bar at 30 °C with a 1% catalyst loading.


Metal: Rh
Ligand type: Phosphine
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Reaction: Hydrogenation
Max TON: ---
ee: 94
PDB: ---
Notes: ---

Artificial Metalloenzymes Through Cysteine-Selective Conjugation of Phosphines to Photoactive Yellow Protein

Kamer, P.C.J.

ChemBioChem 2010, 11, 1236-1239, 10.1002/cbic.201000159

Pinning phosphines on proteins: A method for the cysteine‐selective bioconjugation of phosphines has been developed. The photoactive yellow protein has been site‐selectively functionalized with phosphine ligands and phosphine transition metal complexes to afford artificial metalloenzymes that are active in palladium‐catalysed allylic nucleophilic substitution reactions.


Metal: Pd
Ligand type: Allyl; Phosphine
Anchoring strategy: Covalent
Optimization: Chemical & genetic
Reaction: Allylic amination
Max TON: 45
ee: ---
PDB: 2PHY
Notes: ---

Artificial Metalloproteins Containing Co4O4 Cubane Active Sites

Borovik, A.S.; Don Tilley, T.

J. Am. Chem. Soc. 2018, 140, 2739-2742, 10.1021/jacs.7b13052

Artificial metalloproteins (ArMs) containing Co4O4 cubane active sites were constructed via biotin–streptavidin technology. Stabilized by hydrogen bonds (H-bonds), terminal and cofacial CoIII–OH2 moieties are observed crystallographically in a series of immobilized cubane sites. Solution electrochemistry provided correlations of oxidation potential and pH. For variants containing Ser and Phe adjacent to the metallocofactor, 1e–/1H+ chemistry predominates until pH 8, above which the oxidation becomes pH-independent. Installation of Tyr proximal to the Co4O4 active site provided a single H-bond to one of a set of cofacial CoIII–OH2 groups. With this variant, multi-e–/multi-H+ chemistry is observed, along with a change in mechanism at pH 9.5 that is consistent with Tyr deprotonation. With structural similarities to both the oxygen-evolving complex of photosystem II (H-bonded Tyr) and to thin film water oxidation catalysts (Co4O4 core), these findings bridge synthetic and biological systems for water oxidation, highlighting the importance of secondary sphere interactions in mediating multi-e–/multi-H+ reactivity.


Metal: Co
Ligand type: OAc; Pyridine
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: ---
ee: ---
PDB: 6AUC
Notes: Co-complex in Sav WT

Metal: Co
Ligand type: OAc; Pyridine
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: ---
ee: ---
PDB: 6AUE
Notes: Co-complex in Sav S112Y

Artificial Transfer Hydrogenases Based on the Biotin-(Strept)avidin Technology: Fine Tuning the Selectivity by Saturation Mutagenesis of the Host Protein

Ward, T.R.

J. Am. Chem. Soc. 2006, 128, 8320-8328, 10.1021/ja061580o

Incorporation of biotinylated racemic three-legged d6-piano stool complexes in streptavidin yields enantioselective transfer hydrogenation artificial metalloenzymes for the reduction of ketones. Having identified the most promising organometallic catalyst precursors in the presence of wild-type streptavidin, fine-tuning of the selectivity is achieved by saturation mutagenesis at position S112. This choice for the genetic optimization site is suggested by docking studies which reveal that this position lies closest to the biotinylated metal upon incorporation into streptavidin. For aromatic ketones, the reaction proceeds smoothly to afford the corresponding enantioenriched alcohols in up to 97% ee (R) or 70% (S). On the basis of these results, we suggest that the enantioselection is mostly dictated by CH/π interactions between the substrate and the η6-bound arene. However, these enantiodiscriminating interactions can be outweighed in the presence of cationic residues at position S112 to afford the opposite enantiomers of the product.


Metal: Ir
Ligand type: Amino-sulfonamide; Cp*
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: 96
ee: 80
PDB: ---
Notes: ---

Metal: Rh
Ligand type: Amino-sulfonamide; Cp*
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: 73
ee: 60
PDB: ---
Notes: ---

Metal: Ru
Ligand type: Amino-sulfonamide; Benzene
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: 95
ee: 70
PDB: ---
Notes: ---

Metal: Ru
Ligand type: Amino-sulfonamide; P-cymene
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: 79
ee: 97
PDB: ---
Notes: ---

Artificial Transfer Hydrogenases for the Enantioselective Reduction of Cyclic Imines

Ward, T.R.

Angew. Chem. Int. Ed. 2011, 50, 3026-3029, 10.1002/anie.201007820

Man‐made activity: Introduction of a biotinylated iridium piano stool complex within streptavidin affords an artificial imine reductase (see scheme). Saturation mutagenesis allowed optimization of the activity and the enantioselectivity of this metalloenzyme, and its X‐ray structure suggests that a nearby lysine residue acts as a proton source during the transfer hydrogenation.


Metal: Ir
Ligand type: Amino-sulfonamide; Cp*
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: 4000
ee: 96
PDB: 3PK2
Notes: ---

Metal: Rh
Ligand type: Amino-sulfonamide; Cp*
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: 94
ee: 52
PDB: 3PK2
Notes: ---

Metal: Ru
Ligand type: Amino-sulfonamide; P-cymene
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: 97
ee: 22
PDB: 3PK2
Notes: ---

Metal: Ru
Ligand type: Amino-sulfonamide; Benzene
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: 76
ee: 12
PDB: 3PK2
Notes: ---

Asymmetric δ-Lactam Synthesis with a Monomeric Streptavidin Artificial Metalloenzyme

McNaughton, B.R.; Rovis, T.

J. Am. Chem. Soc. 2019, 141, 4815-4819, 10.1021/jacs.9b01596

Reliable design of artificial metalloenzymes (ArMs) to access transformations not observed in nature remains a long-standing and important challenge. We report that a monomeric streptavidin (mSav) Rh(III) ArM permits asymmetric synthesis of α,β-unsaturated-δ-lactams via a tandem C–H activation and [4+2] annulation reaction. These products are readily derivatized to enantioenriched piperidines, the most common N-heterocycle found in FDA approved pharmaceuticals. Desired δ-lactams are achieved in yields as high as 99% and enantiomeric excess of 97% under aqueous conditions at room temperature. Embedding a Rh cyclopentadienyl (Cp*) catalyst in the active site of mSav results in improved stereocontrol and a 7-fold enhancement in reactivity relative to the isolated biotinylated Rh(III) cofactor. In addition, mSav-Rh outperforms its well-established tetrameric forms, displaying 11–33 times more reactivity.


Metal: Rh
Ligand type: Cp*; OAc
Host protein: Streptavidin (monmeric)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Reaction: Lactam synthesis
Max TON: 33
ee: 97
PDB: ---
Notes: ---

Beyond Iron: Iridium-Containing P450 Enzymes for Selective Cyclopropanations of Structurally Diverse Alkenes

Hartwig, J.F.

ACS Cent. Sci. 2017, 3, 302-308, 10.1021/acscentsci.6b00391

Enzymes catalyze organic transformations with exquisite levels of selectivity, including chemoselectivity, stereoselectivity, and substrate selectivity, but the types of reactions catalyzed by enzymes are more limited than those of chemical catalysts. Thus, the convergence of chemical catalysis and biocatalysis can enable enzymatic systems to catalyze abiological reactions with high selectivity. Recently, we disclosed artificial enzymes constructed from the apo form of heme proteins and iridium porphyrins that catalyze the insertion of carbenes into a C–H bond. We postulated that the same type of Ir(Me)-PIX enzymes could catalyze the cyclopropanation of a broad range of alkenes with control of multiple modes of selectivity. Here, we report the evolution of artificial enzymes that are highly active and highly stereoselective for the addition of carbenes to a wide range of alkenes. These enzymes catalyze the cyclopropanation of terminal and internal, activated and unactivated, electron-rich and electron-deficient, conjugated and nonconjugated alkenes. In particular, Ir(Me)-PIX enzymes derived from CYP119 catalyze highly enantio- and diastereoselective cyclopropanations of styrene with ±98% ee, >70:1 dr, >75% yield, and ∼10,000 turnovers (TON), as well as 1,2-disubstituted styrenes with up to 99% ee, 35:1 dr, and 54% yield. Moreover, Ir(Me)-PIX enzymes catalyze cyclopropanation of internal, unactivated alkenes with up to 99% stereoselectivity, 76% yield, and 1300 TON. They also catalyze cyclopropanation of natural products with diastereoselectivities that are complementary to those attained with standard transition metal catalysts. Finally, Ir(Me)-PIX P450 variants react with substrate selectivity that is reminiscent of natural enzymes; they react preferentially with less reactive internal alkenes in the presence of more reactive terminal alkenes. Together, the studies reveal the suitability of Ir-containing P450s to combine the broad reactivity and substrate scope of transition metal catalysts with the exquisite selectivity of enzymes, generating catalysts that enable reactions to occur with levels and modes of activity and selectivity previously unattainable with natural enzymes or transition metal complexes alone.


Metal: Ir
Ligand type: Methyl; Porphyrin
Host protein: Cytochrome P450 (CYP119)
Anchoring strategy: Metal substitution
Optimization: Chemical & genetic
Reaction: Cyclopropanation
Max TON: 10181
ee: 98
PDB: ---
Notes: Selectivity for cis product (cis/trans = 90:1)

Biosynthesis of a Site-Specific DNA Cleaving Protein

Schultz, P.G.

J. Am. Chem. Soc. 2008, 130, 13194-13195, 10.1021/ja804653f

An E. coli catabolite activator protein (CAP) has been converted into a sequence-specific DNA cleaving protein by genetically introducing (2,2′-bipyridin-5-yl)alanine (Bpy-Ala) into the protein. The mutant CAP (CAP-K26Bpy-Ala) showed comparable binding affinity to CAP-WT for the consensus operator sequence. In the presence of Cu(II) and 3-mercaptopropionic acid, CAP-K26Bpy-Ala cleaves double-stranded DNA with high sequence specificity. This method should provide a useful tool for mapping the molecular details of protein−nucleic acid interactions.


Metal: Cu
Ligand type: Bipyridine
Anchoring strategy: ---
Optimization: Chemical & genetic
Max TON: ---
ee: ---
PDB: ---
Notes: Catabolite activator protein from E. coli

Metal: Fe
Ligand type: Bipyridine
Anchoring strategy: ---
Optimization: Chemical & genetic
Max TON: ---
ee: ---
PDB: ---
Notes: Catabolite activator protein from E. coli

Burkavidin: A Novel Secreted Biotin-Binding Protein from the Human Pathogen Burkholderia Pseudomallei

Creus, M.

Protein Expression Purif. 2011, 77, 131-139, 10.1016/j.pep.2011.01.003

The avidin–biotin technology has many applications, including molecular detection; immobilization; protein purification; construction of supramolecular assemblies and artificial metalloenzymes. Here we present the recombinant expression of novel biotin-binding proteins from bacteria and the purification and characterization of a secreted burkavidin from the human pathogen Burkholderia pseudomallei. Expression of the native burkavidin in Escherichia coli led to periplasmic secretion and formation of a biotin-binding, thermostable, tetrameric protein containing an intra-monomeric disulphide bond. Burkavidin showed one main species as measured by isoelectric focusing, with lower isoelectric point (pI) than streptavidin. To exemplify the potential use of burkavidin in biotechnology, an artificial metalloenzyme was generated using this novel protein-scaffold and shown to exhibit enantioselectivity in a rhodium-catalysed hydrogenation reaction.


Metal: Rh
Ligand type: Diphenylphosphine
Host protein: Burkavidin
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Reaction: Hydrogenation
Max TON: ~110
ee: 65
PDB: ---
Notes: ---