43 publications
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8-Amino-5,6,7,8-tetrahydroquinoline in Iridium(III) Biotinylated Cp* Complex as Artificial Imine Reductase
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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: IrHost protein: Streptavidin (Sav)Anchoring strategy: SupramolecularOptimization: Chemical & geneticNotes: ---
Metal: IrHost protein: Streptavidin (Sav)Anchoring strategy: SupramolecularOptimization: Chemical & geneticNotes: ---
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A Dual Anchoring Strategy for the Localization and Activation of Artificial Metalloenzymes Based on the Biotin−Streptavidin Technology
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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.
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An Artificial Imine Reductase Based on the Ribonuclease S Scaffold
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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.
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An NAD(P)H-Dependent Artificial Transfer Hydrogenase for Multienzymatic Cascades
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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: IrHost protein: Streptavidin (Sav)Anchoring strategy: SupramolecularOptimization: Chemical & geneticNotes: ---
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Aqueous Oxidation of Alcohols Catalyzed by Artificial Metalloenzymes Based on the Biotin–Avidin Technology
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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: RuHost protein: Streptavidin (Sav)Anchoring strategy: SupramolecularOptimization: Chemical & geneticNotes: ---
Metal: RuHost protein: Avidin (Av)Anchoring strategy: SupramolecularOptimization: Chemical & geneticNotes: ---
Metal: RuHost protein: Streptavidin (Sav)Anchoring strategy: SupramolecularOptimization: Chemical & geneticNotes: ---
Metal: RhHost protein: Streptavidin (Sav)Anchoring strategy: SupramolecularOptimization: Chemical & geneticNotes: ---
Metal: IrHost protein: Streptavidin (Sav)Anchoring strategy: SupramolecularOptimization: Chemical & geneticNotes: ---
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Aqueous Phase Transfer Hydrogenation of Aryl Ketones Catalysed by Achiral Ruthenium(II) and Rhodium(III) Complexes and their Papain Conjugates
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Appl. Organomet. Chem. 2013, 27, 6-12, 10.1002/aoc.2929
Several ruthenium and rhodium complexes including 2,2′‐dipyridylamine ligands substituted at the central N atom by an alkyl chain terminated by a maleimide functional group were tested along with a newly synthesized Rh(III) complex of unsubstituted 2,2′‐dipyridylamine as catalysts in the transfer hydrogenation of aryl ketones in neat water with formate as hydrogen donor. All of them except one led to the secondary alcohol products with conversion rates depending on the metal complex. Site‐specific anchoring of the N‐maleimide complexes to the single free cysteine residue of the cysteine endoproteinase papain endowed this protein with transfer hydrogenase properties towards 2,2,2‐trifluoroacetophenone. Quantitative conversions were reached with the Rh‐based biocatalysts, while modest enantioselectivities were obtained in certain reactional conditions.
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A Rhodium Complex-Linked β-Barrel Protein as a Hybrid Biocatalyst for Phenylacetylene Polymerization
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Chem. Commun. 2012, 48, 9756, 10.1039/C2CC35165J
Our group recently prepared a hybrid catalyst containing a rhodium complex, Rh(Cp)(cod), with a maleimide moiety at the peripheral position of the Cp ligand. This compound was then inserted into a β-barrel protein scaffold of a mutant of aponitrobindin (Q96C) via a covalent linkage. The hybrid protein is found to act as a polymerization catalyst and preferentially yields trans-poly(phenylacetylene) (PPA), although the rhodium complex without the protein scaffold normally produces cis PPA.
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Artificial Metalloenzymes Derived from Bovine β-Lactoglobulin for the Asymmetric Transfer Hydrogenation of an Aryl Ketone – Synthesis, Characterization and Catalytic Activity
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Dalton Trans. 2014, 43, 5482-5489, 10.1039/c3dt53253d
Protein hybrids resulting from the supramolecular anchoring to bovine β-lactoglobulin of fatty acid-derived Rh(iii) diimine complexes catalysed the asymmetric transfer hydrogenation of trifluoroacetophenone with up to 32% ee.
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Artificial Metalloenzymes for the Diastereoselective Reduction of NAD+ to NAD2H
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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.
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Artificial Transfer Hydrogenases Based on the Biotin-(Strept)avidin Technology: Fine Tuning the Selectivity by Saturation Mutagenesis of the Host Protein
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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: IrHost protein: Streptavidin (Sav)Anchoring strategy: SupramolecularOptimization: Chemical & geneticNotes: ---
Metal: RhHost protein: Streptavidin (Sav)Anchoring strategy: SupramolecularOptimization: Chemical & geneticNotes: ---
Metal: RuHost protein: Streptavidin (Sav)Anchoring strategy: SupramolecularOptimization: Chemical & geneticNotes: ---
Metal: RuHost protein: Streptavidin (Sav)Anchoring strategy: SupramolecularOptimization: Chemical & geneticNotes: ---
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Artificial Transfer Hydrogenases for the Enantioselective Reduction of Cyclic Imines
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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: IrHost protein: Streptavidin (Sav)Anchoring strategy: SupramolecularOptimization: Chemical & geneticNotes: ---
Metal: RhHost protein: Streptavidin (Sav)Anchoring strategy: SupramolecularOptimization: Chemical & geneticNotes: ---
Metal: RuHost protein: Streptavidin (Sav)Anchoring strategy: SupramolecularOptimization: Chemical & geneticNotes: ---
Metal: RuHost protein: Streptavidin (Sav)Anchoring strategy: SupramolecularOptimization: Chemical & geneticNotes: ---
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Asymmetric δ-Lactam Synthesis with a Monomeric Streptavidin Artificial Metalloenzyme
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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: RhHost protein: Streptavidin (monmeric)Anchoring strategy: SupramolecularOptimization: Chemical & geneticNotes: ---
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Biotinylated Rh(III) Complexes in Engineered Streptavidin for Accelerated Asymmetric C–H Activation
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Science 2012, 338, 500-503, 10.1126/science.1226132
Enzymes provide an exquisitely tailored chiral environment to foster high catalytic activities and selectivities, but their native structures are optimized for very specific biochemical transformations. Designing a protein to accommodate a non-native transition metal complex can broaden the scope of enzymatic transformations while raising the activity and selectivity of small-molecule catalysis. Here, we report the creation of a bifunctional artificial metalloenzyme in which a glutamic acid or aspartic acid residue engineered into streptavidin acts in concert with a docked biotinylated rhodium(III) complex to enable catalytic asymmetric carbon-hydrogen (C–H) activation. The coupling of benzamides and alkenes to access dihydroisoquinolones proceeds with up to nearly a 100-fold rate acceleration compared with the activity of the isolated rhodium complex and enantiomeric ratios as high as 93:7.
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Breaking Symmetry: Engineering Single-Chain Dimeric Streptavidin as Host for Artificial Metalloenzymes
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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.
Notes: Additional PDB: 6S50
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Chemically Engineered Papain as Artificial Formate Dehydrogenase for NAD(P)H Regeneration
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Org. Biomol. Chem. 2011, 9, 5720, 10.1039/c1ob05482a
Organometallic complexes of the general formula [(η6-arene)Ru(N⁁N)Cl]+ and [(η5-Cp*)Rh(N⁁N)Cl]+ where N⁁N is a 2,2′-dipyridylamine (DPA) derivative carrying a thiol-targeted maleimide group, 2,2′-bispyridyl (bpy), 1,10-phenanthroline (phen) or ethylenediamine (en) and arene is benzene, 2-chloro-N-[2-(phenyl)ethyl]acetamide or p-cymene were identified as catalysts for the stereoselective reduction of the enzyme cofactors NAD(P)+ into NAD(P)H with formate as a hydride donor. A thorough comparison of their effectiveness towards NAD+ (expressed as TOF) revealed that the RhIII complexes were much more potent catalysts than the RuII complexes. Within the RuII complex series, both the N⁁N and arene ligands forming the coordination sphere had a noticeable influence on the activity of the complexes. Covalent anchoring of the maleimide-functionalized RuII and RhIII complexes to the cysteine endoproteinase papain yielded hybrid metalloproteins, some of them displaying formate dehydrogenase activity with potentially interesting kinetic parameters.
Notes: TOF = 52.1 h-1 for NAD+
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Chimeric Streptavidins as Host Proteins for Artificial Metalloenzymes
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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.
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Metal: RuLigand type: CarbeneHost protein: Streptavidin (Sav)Anchoring strategy: SupramolecularOptimization: GeneticNotes: RCM, biotinylated Hoveyda-Grubbs second generation catalyst
Metal: ---Ligand type: Biotinylated naphthalenediimidHost protein: Streptavidin (Sav)Anchoring strategy: SupramolecularOptimization: GeneticNotes: No metal
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Computational Insights on an Artificial Imine Reductase Based on the Biotin-Streptavidin Technology
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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.
Notes: Prediction of the enantioselectivity by computational methods.
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Cross-Regulation of an Artificial Metalloenzyme
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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: IrHost protein: Streptavidin (Sav)Anchoring strategy: SupramolecularOptimization: Chemical & geneticNotes: Cross-regulated reduction of the antibiotic enrofloxacin by an ArM.
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Directed Evolution of an Artificial Imine Reductase
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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: IrHost protein: Streptavidin (Sav)Anchoring strategy: SupramolecularOptimization: Chemical & geneticNotes: Salsolidine formation; Sav mutant S112A-N118P-K121A-S122M: (R)-selective
Metal: IrHost protein: Streptavidin (Sav)Anchoring strategy: SupramolecularOptimization: Chemical & geneticNotes: Salsolidine formation; Sav mutant S112R-N118P-K121A-S122M-L124Y: (S)-selective
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Efficient in Situ Regeneration of NADH Mimics by an Artificial Metalloenzyme
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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: IrHost protein: Streptavidin (Sav)Anchoring strategy: SupramolecularOptimization: Chemical & geneticNotes: ArM works in combination with the ene reductase (ER) of the Old Yellow Enzyme family fromThermus scotuductus (TsOYE).
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Enantioselective Transfer Hydrogenation of Ketone Catalysed by Artificial Metalloenzymes Derived from Bovine β-Lactoglobulin
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Chem. Commun. 2012, 48, 11984, 10.1039/c2cc36980j
Artificial metalloproteins resulting from the embedding of half-sandwich Ru(II)/Rh(III) fatty acid derivatives within β-lactoglobulin catalysed the asymmetric transfer hydrogenation of trifluoroacetophenone with modest to good conversions and fair ee's.
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Evaluation of Chemical Diversity of Biotinylated Chiral 1,3-Diamines as a Catalytic Moiety in Artificial Imine Reductase
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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: IrHost protein: Streptavidin (Sav)Anchoring strategy: SupramolecularOptimization: Chemical & geneticNotes: ---
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Expanding the Chemical Diversity in Artificial Imine Reductases Based on the Biotin–Streptavidin Technology
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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: IrHost protein: Streptavidin (Sav)Anchoring strategy: SupramolecularOptimization: Chemical & geneticNotes: ---
Metal: IrHost protein: Streptavidin (Sav)Anchoring strategy: SupramolecularOptimization: Chemical & geneticNotes: ---
Metal: IrHost protein: Streptavidin (Sav)Anchoring strategy: SupramolecularOptimization: Chemical & geneticNotes: ---
Metal: IrLigand type: Cp*Host protein: Streptavidin (Sav)Anchoring strategy: SupramolecularOptimization: Chemical & geneticNotes: ---
Metal: IrHost protein: Streptavidin (Sav)Anchoring strategy: SupramolecularOptimization: Chemical & geneticNotes: ---
Metal: IrHost protein: Streptavidin (Sav)Anchoring strategy: SupramolecularOptimization: Chemical & geneticNotes: ---
Metal: IrHost protein: Streptavidin (Sav)Anchoring strategy: SupramolecularOptimization: Chemical & geneticNotes: ---
Metal: IrHost protein: Streptavidin (Sav)Anchoring strategy: SupramolecularOptimization: Chemical & geneticNotes: ---
Metal: IrHost protein: Streptavidin (Sav)Anchoring strategy: SupramolecularOptimization: Chemical & geneticNotes: ---
Metal: RhHost protein: Streptavidin (Sav)Anchoring strategy: SupramolecularOptimization: Chemical & geneticNotes: ---
Metal: RhHost protein: Streptavidin (Sav)Anchoring strategy: SupramolecularOptimization: Chemical & geneticNotes: ---
Metal: RhHost protein: Streptavidin (Sav)Anchoring strategy: SupramolecularOptimization: Chemical & geneticNotes: ---
Metal: RhLigand type: Cp*Host protein: Streptavidin (Sav)Anchoring strategy: SupramolecularOptimization: Chemical & geneticNotes: ---
Metal: RhHost protein: Streptavidin (Sav)Anchoring strategy: SupramolecularOptimization: Chemical & geneticNotes: ---
Metal: RhHost protein: Streptavidin (Sav)Anchoring strategy: SupramolecularOptimization: Chemical & geneticNotes: ---
Metal: RhHost protein: Streptavidin (Sav)Anchoring strategy: SupramolecularOptimization: Chemical & geneticNotes: ---
Metal: RhHost protein: Streptavidin (Sav)Anchoring strategy: SupramolecularOptimization: Chemical & geneticNotes: ---
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Ferritin Encapsulation of Artificial Metalloenzymes: Engineering a Tertiary Coordination Sphere for an Artificial Transfer Hydrogenase
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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: IrHost protein: Streptavidin (Sav)Anchoring strategy: SupramolecularOptimization: Chemical & geneticNotes: ---
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Fluorescence-Based Assay for the Optimization of the Activity of Artificial Transfer Hydrogenase within a Biocompatible Compartment
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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.
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Genetic Engineering of an Artificial Metalloenzyme for Transfer Hydrogenation of a Self-Immolative Substrate in Escherichia coli’s Periplasm
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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: IrHost protein: Streptavidin (Sav)Anchoring strategy: SupramolecularOptimization: Chemical & geneticNotes: ---
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Genetic Optimization of the Catalytic Efficiency of Artificial Imine Reductases Based on Biotin−Streptavidin Technology
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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.
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High-Level Secretion of Recombinant Full-Length Streptavidin in Pichia Pastoris and its Application to Enantioselective Catalysis
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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.
Notes: Sav expression in E. coli
Notes: Sav expression in P. pastoris
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Human Carbonic Anhydrase II as Host Protein for the Creation of Artificial Metalloenzymes: The Asymmetric Transfer Hydrogenation of Imines
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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: IrHost protein: Human carbonic anhydrase II (hCAII)Anchoring strategy: SupramolecularOptimization: Chemical & geneticNotes: ---
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Immobilization of an Artificial Imine Reductase Within Silica Nanoparticles Improves its Performance
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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.
Notes: Reaction in nanoparticles