36 publications

36 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: ---

A Dual Anchoring Strategy for the Localization and Activation of Artificial Metalloenzymes Based on the Biotin−Streptavidin Technology

Ward, T.R.

J. Am. Chem. Soc. 2013, 135, 5384-5388, 10.1021/ja309974s

Artificial metalloenzymes result from anchoring an active catalyst within a protein environment. Toward this goal, various localization strategies have been pursued: covalent, supramolecular, or dative anchoring. Herein we show that introduction of a suitably positioned histidine residue contributes to firmly anchor, via a dative bond, a biotinylated rhodium piano stool complex within streptavidin. The in silico design of the artificial metalloenzyme was confirmed by X-ray crystallography. The resulting artificial metalloenzyme displays significantly improved catalytic performance, both in terms of activity and selectivity in the transfer hydrogenation of imines. Depending on the position of the histidine residue, both enantiomers of the salsolidine product can be obtained.


Metal: Ir
Ligand type: Amino acid; Cp*
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Genetic
Max TON: 14
ee: 11
PDB: ---
Notes: ---

Metal: Rh
Ligand type: Amino acid; Cp*
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Genetic
Max TON: 100
ee: 79
PDB: ---
Notes: ---

An Artificial Imine Reductase Based on the Ribonuclease S Scaffold

Ward, T.R.

ChemCatChem 2014, 6, 736-740, 10.1002/cctc.201300995

Dative anchoring of a piano‐stool complex within ribonuclease S resulted in an artificial imine reductase. The catalytic performance was modulated upon variation of the coordinating amino acid residues in the S‐peptide. Binding of Cp*Ir (Cp*=C5Me5) to the native active site resulted in good conversions and moderate enantiomeric excess values for the synthesis of salsolidine.


Metal: Ir
Ligand type: Amino acid; Cp*
Host protein: Ribonuclease S
Anchoring strategy: Supramolecular
Optimization: Genetic
Max TON: 4
ee: 18
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 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 the Diastereoselective Reduction of NAD+ to NAD2H

Ward, T.R.

Org. Biomol. Chem. 2015, 13, 357-360, 10.1039/c4ob02071e

Stereoselectively labelled isotopomers of NAD(P)H are highly relevant for mechanistic studies of enzymes which utilize them as redox equivalents.


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

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: ---

Breaking Symmetry: Engineering Single-Chain Dimeric Streptavidin as Host for Artificial Metalloenzymes

Ward, T.R.

J. Am. Chem. Soc. 2019, 141, 15869-15878, 10.1021/jacs.9b06923

The biotin–streptavidin technology has been extensively exploited to engineer artificial metalloenzymes (ArMs) that catalyze a dozen different reactions. Despite its versatility, the homotetrameric nature of streptavidin (Sav) and the noncooperative binding of biotinylated cofactors impose two limitations on the genetic optimization of ArMs: (i) point mutations are reflected in all four subunits of Sav, and (ii) the noncooperative binding of biotinylated cofactors to Sav may lead to an erosion in the catalytic performance, depending on the cofactor:biotin-binding site ratio. To address these challenges, we report on our efforts to engineer a (monovalent) single-chain dimeric streptavidin (scdSav) as scaffold for Sav-based ArMs. The versatility of scdSav as host protein is highlighted for the asymmetric transfer hydrogenation of prochiral imines using [Cp*Ir(biot-p-L)Cl] as cofactor. By capitalizing on a more precise genetic fine-tuning of the biotin-binding vestibule, unrivaled levels of activity and selectivity were achieved for the reduction of challenging prochiral imines. Comparison of the saturation kinetic data and X-ray structures of [Cp*Ir(biot-p-L)Cl]·scdSav with a structurally related [Cp*Ir(biot-p-L)Cl]·monovalent scdSav highlights the advantages of the presence of a single biotinylated cofactor precisely localized within the biotin-binding vestibule of the monovalent scdSav. The practicality of scdSav-based ArMs was illustrated for the reduction of the salsolidine precursor (500 mM) to afford (R)-salsolidine in 90% ee and >17 000 TONs. Monovalent scdSav thus provides a versatile scaffold to evolve more efficient ArMs for in vivo catalysis and large-scale applications.


Metal: Ir
Ligand type: Cp*; Phenanthroline
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Genetic
Max TON: 17000
ee: 98
PDB: 6S4Q
Notes: Additional PDB: 6S50

Chimeric Streptavidins as Host Proteins for Artificial Metalloenzymes

Ward, T.R.; Woolfson, D.N.

ACS Catal. 2018, 8, 1476-1484, 10.1021/acscatal.7b03773

The streptavidin scaffold was expanded with well-structured naturally occurring motifs. These chimeric scaffolds were tested as hosts for biotinylated catalysts as artificial metalloenzymes (ArM) for asymmetric transfer hydrogenation, ring-closing metathesis and anion−π catalysis. The additional second coordination sphere elements significantly influence both the activity and the selectivity of the resulting hybrid catalysts. These findings lead to the identification of propitious chimeric streptavidins for future directed evolution efforts of artificial metalloenzymes.


Metal: Ir
Ligand type: Cp*; Diamine
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Genetic
Max TON: 970
ee: 13
PDB: ---
Notes: ---

Metal: Ir
Ligand type: Cp*; Diamine
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Genetic
Max TON: 158
ee: 82
PDB: ---
Notes: ---

Metal: Ru
Ligand type: Carbene
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Genetic
Reaction: Olefin metathesis
Max TON: 105
ee: ---
PDB: ---
Notes: RCM, biotinylated Hoveyda-Grubbs second generation catalyst

Metal: ---
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Genetic
Reaction: Anion-π catalysis
Max TON: 6
ee: 41
PDB: ---
Notes: No metal

Computational Insights on an Artificial Imine Reductase Based on the Biotin-Streptavidin Technology

Maréchal, J.-D.

ACS Catal. 2014, 4, 833-842, 10.1021/cs400921n

We present a computational study that combines protein–ligand docking, quantum mechanical, and quantum mechanical/molecular mechanical calculations to scrutinize the mechanistic behavior of the first artificial enzyme able to enantioselectively reduce cyclic imines. We applied a novel strategy that allows the characterization of transition state structures in the protein host and their associated reaction paths. Of the most striking results of our investigation is the identification of major conformational differences between the transition state geometries of the lowest energy paths leading to (R)- and (S)-reduction products. The molecular features of (R)- and (S)-transition states highlight distinctive patterns of hydrophobic and polar complementarities between the substrate and the binding site. These differences lead to an activation energy gap that stands in very good agreement with the experimentally determined enantioselectivity. This study sheds light on the mechanism by which transfer hydrogenases operate and illustrates how the change of environment (from homogeneous solution conditions to the asymmetric protein frame) affect the reactivity of the organometallic cofactor. It provides novel insights on the complexity in integrating unnatural organometallic compounds into biological scaffolds. The modeling strategy that we pursued, based on the generation of “pseudo transition state” structures, is computationally efficient and suitable for the discovery and optimization of artificial enzymes. Alternatively, this approach can be applied on systems for which a large conformational sampling is needed to identify relevant transition states.


Metal: Ir
Ligand type: Cp*; Diamine
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Genetic
Max TON: ---
ee: 96
PDB: 3PK2
Notes: Prediction of the enantioselectivity by computational methods.

Cross-Regulation of an Artificial Metalloenzyme

Ward, T.R.

Angew. Chem. Int. Ed. 2017, 56, 10156-10160, 10.1002/anie.201702181

Cross‐regulation of complex biochemical reaction networks is an essential feature of living systems. In a biomimetic spirit, we report on our efforts to program the temporal activation of an artificial metalloenzyme via cross‐regulation by a natural enzyme. In the presence of urea, urease slowly releases ammonia that reversibly inhibits an artificial transfer hydrogenase. Addition of an acid, which acts as fuel, allows to maintain the system out of equilibrium.


Metal: Ir
Ligand type: Cp*; Phenanthroline
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: 96
ee: ---
PDB: ---
Notes: Cross-regulated reduction of the antibiotic enrofloxacin by an ArM.

Directed Evolution of an Artificial Imine Reductase

Maréchal, J.-D.; Ward, T.R.

Angew. Chem. Int. Ed. 2018, 57, 1863-1868, 10.1002/anie.201711016

Artificial metalloenzymes, resulting from incorporation of a metal cofactor within a host protein, have received increasing attention in the last decade. The directed evolution is presented of an artificial transfer hydrogenase (ATHase) based on the biotin‐streptavidin technology using a straightforward procedure allowing screening in cell‐free extracts. Two streptavidin isoforms were yielded with improved catalytic activity and selectivity for the reduction of cyclic imines. The evolved ATHases were stable under biphasic catalytic conditions. The X‐ray structure analysis reveals that introducing bulky residues within the active site results in flexibility changes of the cofactor, thus increasing exposure of the metal to the protein surface and leading to a reversal of enantioselectivity. This hypothesis was confirmed by a multiscale approach based mostly on molecular dynamics and protein–ligand dockings.


Metal: Ir
Ligand type: Amino-sulfonamide; Cp*
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: 380
ee: 95
PDB: 6ESS
Notes: Salsolidine formation; Sav mutant S112A-N118P-K121A-S122M: (R)-selective

Metal: Ir
Ligand type: Amino-sulfonamide; Cp*
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: 220
ee: 85
PDB: 6ESU
Notes: Salsolidine formation; Sav mutant S112R-N118P-K121A-S122M-L124Y: (S)-selective

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: ---

Expanding the Chemical Diversity in Artificial Imine Reductases Based on the Biotin–Streptavidin Technology

Ward, T.R.

ChemCatChem 2014, 6, 1010-1014, 10.1002/cctc.201300825

We report on the optimization of an artificial imine reductase based on the biotin‐streptavidin technology. With the aim of rapidly generating chemical diversity, a novel strategy for the formation and evaluation of biotinylated complexes is disclosed. Tethering the biotin‐anchor to the Cp* moiety leaves three free coordination sites on a d6 metal for the introduction of chemical diversity by coordination of a variety of ligands. To test the concept, 34 bidentate ligands were screened and a selection of the 6 best was tested in the presence of 21 streptavidin (Sav) isoforms for the asymmetric imine reduction by the resulting three legged piano stool complexes. Enantiopure α‐amino amides were identified as promising bidentate ligands: up to 63 % ee and 190 turnovers were obtained in the formation of 1‐phenyl‐1,2,3,4‐tetrahydroisoquinoline with [IrCp*biotin(L‐ThrNH2)Cl]⊂SavWT as a catalyst.


Metal: Ir
Ligand type: Amino acid; Cp*
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: 188
ee: 43
PDB: ---
Notes: ---

Metal: Ir
Ligand type: Amino carboxylic acid; Cp*
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: 4
ee: 21
PDB: ---
Notes: ---

Metal: Ir
Ligand type: Cp*; Diamine
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: 0
ee: ---
PDB: ---
Notes: ---

Metal: Ir
Ligand type: Cp*
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: 0
ee: ---
PDB: ---
Notes: ---

Metal: Ir
Ligand type: Cp*; Pyrazine amide
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: 26
ee: 16
PDB: ---
Notes: ---

Metal: Ir
Ligand type: Bipyridine; Cp*
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: 0
ee: ---
PDB: ---
Notes: ---

Metal: Ir
Ligand type: Amino-sulfonamide; Cp*
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: 12
ee: 13
PDB: ---
Notes: ---

Metal: Ir
Ligand type: Cp*; Oxazoline
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: 102
ee: 14
PDB: ---
Notes: ---

Metal: Ir
Ligand type: Amino acid; Cp*
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: 94
ee: 67
PDB: ---
Notes: ---

Metal: Rh
Ligand type: Amino amide; Cp*
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: 10
ee: 7
PDB: ---
Notes: ---

Metal: Rh
Ligand type: Amino carboxylic acid; Cp*
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: 8
ee: 1
PDB: ---
Notes: ---

Metal: Rh
Ligand type: Cp*; Diamine
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: 6
ee: 1
PDB: ---
Notes: ---

Metal: Rh
Ligand type: Cp*
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: 6
ee: 1
PDB: ---
Notes: ---

Metal: Rh
Ligand type: Cp*; Pyrazine amide
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: 6
ee: 1
PDB: ---
Notes: ---

Metal: Rh
Ligand type: Bipyridine; Cp*
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: 4
ee: 6
PDB: ---
Notes: ---

Metal: Rh
Ligand type: Amino-sulfonamide; Cp*
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: 6
ee: 1
PDB: ---
Notes: ---

Metal: Rh
Ligand type: Cp*; Oxazoline
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: 8
ee: 0
PDB: ---
Notes: ---

Ferritin Encapsulation of Artificial Metalloenzymes: Engineering a Tertiary Coordination Sphere for an Artificial Transfer Hydrogenase

Ward, T.R.

Dalton Trans. 2018, 47, 10837-10841, 10.1039/C8DT02224K

Ferritin, a naturally occuring iron-storage protein, plays an important role in nanoengineering and biomedical applications. Upon iron removal, apoferritin was shown to allow the encapsulation of an artificial transfer hydrogenase (ATHase) based on the streptavidin-biotin technology. The third coordination sphere, provided by ferritin, significantly influences the catalytic activity of an ATHase for the reduction of cyclic imines.


Metal: Ir
Ligand type: Amino-sulfonamide; Cp*
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: 3874
ee: 75
PDB: ---
Notes: ---

Fluorescence-Based Assay for the Optimization of the Activity of Artificial Transfer Hydrogenase within a Biocompatible Compartment

Ward, T.R.

ChemCatChem 2013, 5, 720-723, 10.1002/cctc.201200834

The time capsules: The transfer hydrogenation of an enone‐bound fluorogenic compound by an artificial metalloenzyme leads to the release of fluorescent compound umbelliferone. Upon encapsulation of the hybrid catalyst inside a biocompatible compartment, the activity of the transfer hydrogenase is maintained for several months, even at micromolar concentrations.


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

Genetic Engineering of an Artificial Metalloenzyme for Transfer Hydrogenation of a Self-Immolative Substrate in Escherichia coli’s Periplasm

Ward, T.R.

J. Am. Chem. Soc. 2018, 140, 13171-13175, 10.1021/jacs.8b07189

Artificial metalloenzymes (ArMs), which combine an abiotic metal cofactor with a protein scaffold, catalyze various synthetically useful transformations. To complement the natural enzymes’ repertoire, effective optimization protocols to improve ArM’s performance are required. Here we report on our efforts to optimize the activity of an artificial transfer hydrogenase (ATHase) using Escherichia coli whole cells. For this purpose, we rely on a self-immolative quinolinium substrate which, upon reduction, releases fluorescent umbelliferone, thus allowing efficient screening. Introduction of a loop in the immediate proximity of the Ir-cofactor afforded an ArM with up to 5-fold increase in transfer hydrogenation activity compared to the wild-type ATHase using purified mutants.


Metal: Ir
Ligand type: Amino-sulfonamide; Cp*
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: 1000
ee: 76
PDB: 6GMI
Notes: ---

Genetic Optimization of the Catalytic Efficiency of Artificial Imine Reductases Based on Biotin−Streptavidin Technology

Ward, T.R.

ACS Catal. 2013, 3, 1752-1755, 10.1021/cs400428r

Artificial metalloenzymes enable the engineering of the reaction microenvironment of the active metal catalyst by modification of the surrounding host protein. We report herein the optimization of an artificial imine reductase (ATHase) based on biotin–streptavidin technology. By introduction of lipophilic amino acid residues around the active site, an 8-fold increase in catalytic efficiency compared with the wild type imine reductase was achieved. Whereas substrate inhibition was encountered for the free cofactor and wild type ATHase, two engineered systems exhibited classical Michaelis–Menten kinetics, even at substrate concentrations of 150 mM with measured rates up to 20 min–1.


Metal: Ir
Ligand type: Amino-sulfonamide; Cp*
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Genetic
Max TON: ---
ee: 60
PDB: ---
Notes: ---

High-Level Secretion of Recombinant Full-Length Streptavidin in Pichia Pastoris and its Application to Enantioselective Catalysis

Jaussi, R.

Protein Expression Purif. 2014, 93, 54-62, 10.1016/j.pep.2013.10.015

Artificial metalloenzymes result from the incorporation of a catalytically competent biotinylated organometallic moiety into full-length (i.e. mature) streptavidin. With large-scale industrial biotechnology applications in mind, large quantities of recombinant streptavidin are required. Herein we report our efforts to produce wild-type mature and biotin-free streptavidin using the yeast Pichia pastoris expression system. The streptavidin gene was inserted into the expression vector pPICZαA in frame with the Saccharomyces cerevisiae α-mating factor secretion signal. In a fed-batch fermentation using a minimal medium supplemented with trace amounts of biotin, functional streptavidin was secreted at approximately 650 mg/L of culture supernatant. This yield is approximately threefold higher than that from Escherichia coli, and although the overall expression process takes longer (ten days vs. two days), the downstream processing is simplified by eliminating denaturing/refolding steps. The purified streptavidin bound ∼3.2 molecules of biotin per tetramer. Upon incorporation of a biotinylated piano-stool catalyst, the secreted streptavidin displayed identical properties to streptavidin produced in E. coli by showing activity as artificial imine reductase.


Metal: Ir
Ligand type: Amino-sulfonamide; Cp*
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Genetic
Max TON: 152
ee: 61
PDB: ---
Notes: Sav expression in E. coli

Metal: Ir
Ligand type: Amino-sulfonamide; Cp*
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Genetic
Max TON: 158
ee: 64
PDB: ---
Notes: Sav expression in P. pastoris

Human Carbonic Anhydrase II as Host Protein for the Creation of Artificial Metalloenzymes: The Asymmetric Transfer Hydrogenation of Imines

Ward, T.R.

Chem. Sci. 2013, 4, 3269, 10.1039/c3sc51065d

In the presence of human carbonic anhydrase II, aryl-sulfonamide-bearing IrCp* pianostool complexes catalyze the asymmetric transfer hydrogenation of imines. Critical cofactor–protein interactions revealed by the X-ray structure of [(η5-Cp*)Ir(pico 4)Cl] 9 ⊂ WT hCA II were genetically optimized to improve the catalytic performance of the artificial metalloenzyme (68% ee, kcat/KM 6.11 × 10−3 min−1 mM−1).


Metal: Ir
Ligand type: Amino-sulfonamide; Cp*
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: 47
ee: 70
PDB: ---
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

Improving the Catalytic Performance of an Artificial Metalloenzyme by Computational Design

Baker, D.; Ward, T.R.

J. Am. Chem. Soc. 2015, 137, 10414-10419, 10.1021/jacs.5b06622

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), [(η5-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 [(η5-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.


Metal: Ir
Ligand type: Cp*; Pyridine sulfonamide
Anchoring strategy: Supramolecular
Optimization: Genetic
Max TON: 100
ee: 96
PDB: ---
Notes: ---

Improving the Enantioselectivity of Artificial Transfer Hydrogenases Based on the Biotin–Streptavidin Technology by Combinations of Point Mutations

Ward, T.R.

Inorg. Chim. Acta 2010, 363, 601-604, 10.1016/j.ica.2009.02.001

Artificial metalloenzymes based on the incorporation of biotinylated ruthenium piano–stool complexes within streptavidin can be readily optimized by chemical or genetic means. We performed genetic modifications of such artificial metalloenzymes for the transfer hydrogenation of aromatic ketones, by combining targeted point mutations of the host protein. Upon using the P64G-L124V double mutant of streptavidin in combination with the [η6-(p-cymene)Ru(Biot-p-L)Cl] complex, the enantioselectivity can be increased up to 98% ee (R) for the reduction of p-methylacetophenone, which is the highest selectivity obtained up to date with an artificial transfer hydrogenase.


Metal: Ru
Ligand type: Amino-sulfonamide; P-cymene
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: 98
ee: 98
PDB: ---
Notes: ---

Metal: Ru
Ligand type: Amino-sulfonamide; Benzene
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: 24
ee: 84
PDB: 2QCB
Notes: ---

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)

Neutralizing the Detrimental Effect of Glutathione on Precious Metal Catalysts

Ward, T.R.

J. Am. Chem. Soc. 2014, 136, 8928-8932, 10.1021/ja500613n

We report our efforts to enable transition-metal catalysis in the presence of cellular debris, notably Escherichia coli cell free extracts and cell lysates. This challenging goal is hampered by the presence of thiols, mainly present in the form of glutathione (GSH), which poison precious metal catalysts. To overcome this, we evaluated a selection of oxidizing agents and electrophiles toward their potential to neutralize the detrimental effect of GSH on a Ir-based transfer hydrogenation catalyst. While the bare catalyst was severely inhibited by cellular debris, embedding the organometallic moiety within a host protein led to promising results in the presence of some neutralizing agents. In view of its complementary to natural enzymes, the asymmetric imine reductase based on the incorporation of a biotinylated iridium pianostool complex within streptavidin (Sav) isoforms was selected as a model reaction. Compared to purified protein samples, we show that pretreatment of cell free extracts and cell lysates containing Sav mutants with diamide affords up to >100 TON’s and only a modest erosion of enantioselectivity.


Metal: Ir
Ligand type: Amino-sulfonamide; Cp*
Host protein: Streptavidin (Sav)
Anchoring strategy: Supramolecular
Optimization: Chemical & genetic
Max TON: 98
ee: 85
PDB: ---
Notes: Reaction in cell-free extract with diamide

Piano-Stool d(6)-Rhodium(III) Complexes of Chelating Pyridine-Based Ligands and their Papain Bioconjugates for the Catalysis of Transfer Hydrogenation of Aryl Ketones in Aqueous Medium

Mangiatordi, G.F.; Salmain, M.

J. Mol. Catal. B: Enzym. 2015, 122, 314-322, 10.1016/j.molcatb.2015.10.007

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 24 h 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.


Metal: Rh
Ligand type: Cp*; Phenanthroline
Host protein: Papain (PAP)
Anchoring strategy: Covalent
Optimization: Chemical
Max TON: 30
ee: 9
PDB: ---
Notes: ---

Metal: Rh
Ligand type: Cp*; Di(2-pyridyl)
Host protein: Papain (PAP)
Anchoring strategy: Covalent
Optimization: Chemical
Max TON: 20
ee: 5
PDB: ---
Notes: ---

Porous Protein Crystals as Catalytic Vessels for Organometallic Complexes

Kitagawa, S.; Ueno, T.

Chem. - Asian J. 2014, 9, 1373-1378, 10.1002/asia.201301347

Porous protein crystals, which are protein assemblies in the solid state, have been engineered to form catalytic vessels by the incorporation of organometallic complexes. Ruthenium complexes in cross‐linked porous hen egg white lysozyme (HEWL) crystals catalyzed the enantioselective hydrogen‐transfer reduction of acetophenone derivatives. The crystals accelerated the catalytic reaction and gave different enantiomers based on the crystal form (tetragonal or orthorhombic). This method represents a new approach for the construction of bioinorganic catalysts from protein crystals.


Metal: Ru
Ligand type: Benzene
Host protein: Lysozyme (crystal)
Anchoring strategy: Dative
Optimization: ---
Max TON: ---
ee: ---
PDB: 3W6A
Notes: Tetragonal HEWL crystals

Metal: Ru
Ligand type: Benzene
Host protein: Lysozyme (crystal)
Anchoring strategy: Dative
Optimization: ---
Max TON: ---
ee: ---
PDB: 4J7V
Notes: Orthorhombic HEWL crystals

Proteins as Macromolecular Ligands for Metal-Catalysed Asymmetric Transfer Hydrogenation of Ketones in Aqueous Medium

Salmain, M.

Eur. J. Inorg. Chem. 2018, 2018, 1383-1393, 10.1002/ejic.201701359

Biohybrid catalysts resulting from the dative anchoring of half‐sandwich organometallic complexes [M(arene)(H2O)x(Cl)y]n+ (M = RuII, arene = η6‐benzene, p‐cymene or mesitylene; M = IrIII, RhIII, arene = η5‐Cp*; x = 1–3, y = 0–2, n = 0–2) to bovine beta‐lactoglobulin (βLG) or hen egg white lysozyme showed unprecedented behaviour. These constructs were shown to catalyse the asymmetric transfer hydrogenation of aryl ketones in water with sodium formate as hydrogen donor at a much faster rate than the complexes alone. Full conversion of the benchmark substrate 2,2,2‐trifluoroacetophenone was reached with an ee of 86 % for the most selective biohybrid. Surprisingly, even the crude biohybrid gave a good ee despite the presence of non‐protein‐bound metal species in the reaction medium. Other aryl ketones were reduced in the same way, and the highest ee was obtained for ortho‐substituted acetophenone derivatives. Furthermore, treatment of βLG with dimethyl pyrocarbonate resulted in a noticeable decrease of the activity and selectivity of the biohybrid, indicating that the sole accessible histidine residue (His146) was probably involved in the coordination and activation of Ru(benzene). This work underscores that protein scaffolds are efficient chiral ligands for asymmetric catalysis. The use of sodium formate instead of dihydrogen makes this approach safe, inexpensive and environmentally friendly.


Metal: Ru
Ligand type: Benzene derivatives
Anchoring strategy: Undefined
Optimization: ---
Max TON: 43
ee: 82
PDB: ---
Notes: ---

Metal: Rh
Ligand type: Cp*
Anchoring strategy: Undefined
Optimization: ---
Max TON: 16
ee: 14
PDB: ---
Notes: ---

Metal: Ir
Ligand type: Cp*
Anchoring strategy: Undefined
Optimization: ---
Max TON: 20
ee: 16
PDB: ---
Notes: ---