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.

Hartwig, J.F.

Nat. Chem. 2021, 13, 312-318, 10.1038/s41557-020-00633-7

Enzymatic reactions through mononuclear metal hydrides are unknown in nature, despite the prevalence of such intermediates in the reactions of synthetic transition-metal catalysts. If metalloenzymes could react through abiotic intermediates like these, then the scope of enzyme-catalysed reactions would expand. Here we show that zinc-containing carbonic anhydrase enzymes catalyse hydride transfers from silanes to ketones with high enantioselectivity. We report mechanistic data providing strong evidence that the process involves a mononuclear zinc hydride. This work shows that abiotic silanes can act as reducing equivalents in an enzyme-catalysed process and that monomeric hydrides of electropositive metals, which are typically unstable in protic environments, can be catalytic intermediates in enzymatic processes. Overall, this work bridges a gap between the types of transformation in molecular catalysis and biocatalysis.

Renaud, J.-L.; Ward, T.R.

Catal. Lett. 2016, 146, 564-569, 10.1007/s10562-015-1681-6

We report on the synthesis of biotinylated (cyclopentadienone)iron tricarbonyl complexes, the in situ generation of the corresponding streptavidin conjugates and their application in asymmetric hydrogenation of imines and ketones.

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.

Akabori, S.; Sakurai, S.

Nature 1956, 178, 323-324, 10.1038/178323b0

Asymmetric synthesis has hitherto succeeded only by using reagents or solvents having the asymmetric configuration.

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.

Shaw, W.J.

Organometallics 2020, 39, 1532-1544, 10.1021/acs.organomet.9b00843

The protein scaffold around the active site of enzymes is known to influence catalytic activity, but specific scaffold features responsible for favorable influences are often not known. This study focuses on using an artificial metalloenzyme to probe one specific feature of the scaffold, the position of a positive charge in the outer coordination sphere around the active site. Previous work showed that a small molecular complex, [Rh(PEt2NglycinePEt2)2]−, immobilized covalently within a protein scaffold was activated for CO2 hydrogenation. Here, using an iterative design where the effect of arginine, histidine, or lysine residues placed in the outer coordination sphere of the catalytic active site were evaluated, we tested the hypothesis that positively charged groups facilitate CO2 hydrogenation with seven unique constructs. Single-, double-, and triple-point mutations were introduced to directly compare catalytic activity, as monitored by turnover frequencies (TOFs) measured in real time with 1H NMR spectroscopy, and evaluate related structural and electronic properties. Two of the seven constructs showed a 2- and 3-fold increase relative to the wild type, with overall rates ranging from 0.2 to 0.7 h–1, and a crystal structure of the fastest of these shows the positive charge positioned next to the active site. A crystal structure of the arginine-containing complex shows that the arginines are positioned near the metal. Molecular dynamics (MD) studies also suggest that the positive charge is oriented next to the active site in the two constructs with faster rates but not in the others and that the positive charge near the active site holds the CO2 near the metal, all consistent with a positive charge appropriately positioned in the scaffold benefiting catalysis. The MD studies also suggest that changes in the water distribution around the active site may contribute to catalytic activity, while modest structural changes and movement of the complex within the scaffold do not.

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.

Mahy, J.-P.

Biotechnol. Appl. Biochem. 2020, 67, 563-573, 10.1002/bab.1906

We report a new artificial hydrogenase made by covalent anchoring of the iron Knölker's complex to a xylanase S212C variant. This artificial metalloenzyme was found to be able to catalyze efficiently the transfer hydrogenation of the benchmark substrate trifluoroacetophenone by sodium formate in water, yielding the corresponding secondary alcohol as a racemic. The reaction proceeded more than threefold faster with the XlnS212CK biohybrid than with the Knölker's complex alone. In addition, efficient conversion of trifluoroacetophenone to its corresponding alcohol was reached within 60 H with XlnS212CK, whereas a ≈2.5-fold lower conversion was observed with Knölker's complex alone as catalyst. Moreover, the data were rationalized with a computational strategy suggesting the key factors of the selectivity. These results suggested that the Knölker's complex was most likely flexible and could experience free rotational reorientation within the active-site pocket of Xln A, allowing it to access the subsite pocket populated by trifluoroacetophenone.

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.

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.

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

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

Ward, T.R.

J. Organomet. Chem. 2004, 689, 4868-4871, 10.1016/j.jorganchem.2004.09.032

We report on the phenomenon of protein-accelerated catalysis in the field of artificial metalloenzymes based on the non-covalent incorporation of biotinylated rhodium–diphosphine complexes in (strept)avidin as host proteins. By incrementally varying the [Rh(COD)(Biot-1)]+ vs. (strept)avidin ratio, we show that the enantiomeric excess of the produced acetamidoalanine decreases slowly. This suggests that the catalyst inside (strept)avidin is more active than the catalyst outside the host protein. Both avidin and streptavidin display protein-accelerated catalysis as the protein embedded catalyst display 12.0- and 3.0-fold acceleration over the background reaction with a catalyst devoid of protein. Thus, these artificial metalloenzymes display an increase both in activity and in selectivity for the reduction of acetamidoacrylic acid.

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. 2004, 126, 14411-14418, 10.1021/ja0476718

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

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.

Harada, A.

Org. Biomol. Chem. 2006, 4, 3571, 10.1039/B609242J

Monoclonal antibodies have been elicited against an achiral rhodium complex and this complex was used in the presence of a resultant antibody, 1G8, for the catalytic hydrogenation of 2-acetamidoacrylic acid to produce N-acetyl-L-alanine in high (>98%) enantiomeric excess.

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.

Creus, M.

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

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

Chan, A.S.C.

Tetrahedron: Asymmetry 1999, 10, 1887-1893, 10.1016/S0957-4166(99)00193-7

The construction of a chiral catalyst system embedded at a specific site in a protein has been studied. The preparation of the biotinylated Pyrphos–Rh(I) complex attached to the binding site in avidin and its application to the asymmetric hydrogenation of itaconic acid have been investigated. By introducing the chiral Pyrphos–Rh(I) moiety into the constrained environment of the protein cavity it was found that the enantioselectivity of the system was significantly influenced by the tertiary conformation within the avidin cavity. The effects of reaction conditions such as temperature, hydrogen pressure, and the pH value of the buffer on enantioselectivity are reported.

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.

Chem. Commun. 2005, 4815, 10.1039/b509015f

Incorporation of biotinylated-[rhodium(diphosphine)]+ complexes, with enantiopure amino acid spacers, in streptavidin affords solvent-tolerant and selective artificial metalloenzymes: up to 91% ee (S) in the hydrogenation of N-protected dehydroamino acids.

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.

Barker, P.D.; Boss, S.R.

Angew. Chem. Int. Ed. 2021, 60, 10919-10927, 10.1002/anie.202015834

Many natural metalloenzymes assemble from proteins and biosynthesised complexes, generating potent catalysts by changing metal coordination. Here we adopt the same strategy to generate artificial metalloenzymes (ArMs) using ligand exchange to unmask catalytic activity. By systematically testing RuII(η6-arene)(bipyridine) complexes designed to facilitate the displacement of functionalised bipyridines, we develop a fast and robust procedure for generating new enzymes via ligand exchange in a protein that has not evolved to bind such a complex. The resulting metal cofactors form peptidic coordination bonds but also retain a non-biological ligand. Tandem mass spectrometry and 19F NMR spectroscopy were used to characterise the organometallic cofactors and identify the protein-derived ligands. By introduction of ruthenium cofactors into a 4-helical bundle, transfer hydrogenation catalysts were generated that displayed a 35-fold rate increase when compared to the respective small molecule reaction in solution.

Whitesides, G.M.

J. Am. Chem. Soc. 1978, 100, 306-307, 10.1021/ja00469a064

n/a