6 publications

6 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: ---

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.

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).

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: ---

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)