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.

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.

Pellegrino, S.; Rimoldi, I.

Inorg. Chem. 2021, 60, 2976-2982, 10.1021/acs.inorgchem.0c02969

Based on the supramolecular interaction between vancomycin (Van), an antibiotic glycopeptide, and D-Ala-D-Ala (DADA) dipeptides, a novel class of artificial metalloenzymes was synthesized and characterized. The presence of an iridium(III) ligand at the N-terminus of DADA allowed the use of the metalloenzyme as a catalyst in the asymmetric transfer hydrogenation of cyclic imines. In particular, the type of link between DADA and the metal-chelating moiety was found to be fundamental for inducing asymmetry in the reaction outcome, as highlighted by both computational studies and catalytic results. Using the [IrCp*(m-I)Cl]Cl ⊂ Van complex in 0.1 M CH3COONa buffer at pH 5, a significant 70% (S) e.e. was obtained in the reduction of quinaldine B.

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.

Okuda, J.

Org. Biomol. Chem. 2021, 19, 2912-2916, 10.1039/d1ob00279a

A modified Cp*Ru complex, equipped with a maleimide group, was covalently attached to a cysteine of an engineered variant of Ferric hydroxamate uptake protein component: A (FhuA). This synthetic metalloprotein catalyzed the intermolecular alkene–alkyne coupling of 3-butenol with 5-hexynenitrile. When compared with the protein-free Cp*Ru catalyst, the biohybrid catalyst produced the linear product with higher regioselectivity.

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.

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.

Salmain, M.

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.

Hayashi, T

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.

Salmain, M.

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.

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.

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.

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.

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.

Rovis, T.; Ward, T.R.

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.

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.

Salmain, M.

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.

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.

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.

Jäger, C.M.; Pordea, A.

Faraday Discuss. 2022, 10.1039/d1fd00070e

Artificial metalloenzymes (ArMs) confer non-biological reactivities to biomolecules, whilst taking advantage of the biomolecular architecture in terms of their selectivity and renewable origin. In particular, the design of ArMs by the supramolecular anchoring of metal catalysts to protein hosts provides flexible and easy to optimise systems. The use of cofactor dependent enzymes as hosts gives the advantage of both a (hydrophobic) binding site for the substrate and a cofactor pocket to accommodate the catalyst. Here, we present a computationally driven design approach of ArMs for the transfer hydrogenation reaction of cyclic imines, starting from the NADP+-dependent alcohol dehydrogenase from Thermoanaerobacter brockii (TbADH). We tested and developed a molecular docking workflow to define and optimize iridium catalysts with high affinity for the cofactor binding site of TbADH. The workflow uses high throughput docking of compound libraries to identify key structural motifs for high affinity, followed by higher accuracy docking methods on smaller, focused ligand and catalyst libraries. Iridium sulfonamide catalysts were selected and synthesised, containing either a triol, a furane, or a carboxylic acid to provide the interaction with the cofactor binding pocket. IC50 values of the resulting complexes during TbADH-catalysed alcohol oxidation were determined by competition experiments and were between 4.410 mM and 0.052 mM, demonstrating the affinity of the iridium complexes for either the substrate or the cofactor binding pocket of TbADH. The catalytic activity of the free iridium complexes in solution showed a maximal turnover number (TON) of 90 for the reduction of salsolidine by the triol-functionalised iridium catalyst, whilst in the presence of TbADH, only the iridium catalyst with the triol anchoring functionality showed activity for the same reaction (TON of 36 after 24 h). The observation that the artificial metalloenzymes developed here lacked stereoselectivity demonstrates the need for the further investigation and optimisation of the ArM. Our results serve as a starting point for the design of robust artificial metalloenzymes, exploiting supramolecular anchoring to natural NAD(P)H binding pockets.

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.

Pordea, A.

J. Inorg. Biochem. 2021, 220, 111446, 10.1016/j.jinorgbio.2021.111446

Artificial metalloenzymes result from the insertion of a catalytically active metal complex into a biological scaffold, generally a protein devoid of other catalytic functionalities. As such, their design requires efforts to engineer substrate binding, in addition to accommodating the artificial catalyst. Here we constructed and characterised artificial metalloenzymes using alcohol dehydrogenase as starting point, an enzyme which has both a cofactor and a substrate binding pocket. A docking approach was used to determine suitable positions for catalyst anchoring to single cysteine mutants, leading to an artificial metalloenzyme capable to reduce both natural cofactors and the hydrophobic 1-benzylnicotinamide mimic. Kinetic studies revealed that the new construct displayed a Michaelis-Menten behaviour with the native nicotinamide cofactors, which were suggested by docking to bind at a surface exposed site, different compared to their native binding position. On the other hand, the kinetic and docking data suggested that a typical enzyme behaviour was not observed with the hydrophobic 1-benzylnicotinamide mimic, with which binding events were plausible both inside and outside the protein. This work demonstrates an extended substrate scope of the artificial metalloenzymes and provides information about the binding sites of the nicotinamide substrates, which can be exploited to further engineer artificial metalloenzymes for cofactor regeneration.

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.

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.

Hromadová, M.

J. Electroanal. Chem. 2020, 859, 113882, 10.1016/j.jelechem.2020.113882

Electrochemical properties were studied for [(η6-arene)Ru(1,10-phenanthroline)Cl]Cl (arene = C6H5(CH2)2NHCOCH2Cl) organometallic complex 1, protein Papain PAP and its conjugate with organometallic complex 1-PAP. The latter can serve as an artificial metalloenzyme with catalytic activity in transfer hydrogenation. This work demonstrates that AC voltammetry and electrochemical impedance spectroscopy can be used as fast tools to screen the catalytic ability of 1-PAP electrochemically by studies of the catalytic hydrogen evolution reaction (HER). Proteins are known to catalyze this process, but we have shown that additional HER signal associated with the catalytic activity of 1 is observed for its conjugate with Papain 1-PAP.

Salmain, M.

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.

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.

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.

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.

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.