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.

Lewis, J.C.

ChemBioChem 2014, 15, 223-227, 10.1002/cbic.201300661

Strain‐promoted azide–alkyne cycloaddition (SPAAC) can be used to generate artificial metalloenzymes (ArMs) from scaffold proteins containing a p‐azido‐L‐phenylalanine (Az) residue and catalytically active bicyclononyne‐substituted metal complexes. The high efficiency of this reaction allows rapid ArM formation when using Az residues within the scaffold protein in the presence of cysteine residues or various reactive components of cellular lysate. In general, cofactor‐based ArM formation allows the use of any desired metal complex to build unique inorganic protein materials. SPAAC covalent linkage further decouples the native function of the scaffold from the installation process because it is not affected by native amino acid residues; as long as an Az residue can be incorporated, an ArM can be generated. We have demonstrated the scope of this method with respect to both the scaffold and cofactor components and established that the dirhodium ArMs generated can catalyze the decomposition of diazo compounds and both SiH and olefin insertion reactions involving these carbene precursors.

Ward, T.R.

Cat. Sci. Technol. 2018, 8, 2294-2298, 10.1039/C8CY00646F

We report an artificial carbenoid transferase which combines a biotinylated dirhodium moiety within streptavidin scaffold.

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.

Marchetti, M.

Tetrahedron Lett. 2000, 41, 3717-3720, 10.1016/S0040-4039(00)00473-1

A highly efficient and chemoselective biphasic hydroformylation of olefins was accomplished using water soluble complexes formed by the interaction between Rh(CO)2(acac) and human serum albumin (HSA), a readily available water soluble protein. A new type of shape-selectivity was observed in the hydroformylation of sterically hindered olefins.

Marchetti, M.

Adv. Synth. Catal. 2002, 344, 556, 10.1002/1615-4169(200207)344:5<556::AID-ADSC556>3.0.CO;2-E

The water‐soluble complex derived from Rh(CO)2(acac) and human serum albumin (HSA) proved to be efficient in the hydroformylation of several olefin substrates. The chemoselectivity and regioselectivity were generally higher than those obtained by using the classic catalytic systems like TPPTS‐Rh(I) (TPPTS=triphenylphosphine‐3,3′,3″‐trisulfonic acid trisodium salt). Styrene and 1‐octene, for instance, were converted in almost quantitative yields into the corresponding oxo‐aldehydes at 60 °C and 70 atm (CO/H2=1) even at very low Rh(CO)2(acac)/HSA catalyst concentrations. The possibility of easily recovering the Rh(I) compound makes the system environmentally friendly. The circular dichroism technique was useful for demonstrating the Rh(I) binding to the protein and to give information on the stability in solution of the catalytic system. Some other proteins have been used to replace HSA as complexing agent for Rh(I). The results were less impressive than those obtained using HSA and their complexes with Rh(I) were much less stable.

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.

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.

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.

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.

Schwaneberg, U.

ACS Catal. 2018, 8, 2611-2614, 10.1021/acscatal.7b04369

Whole cell catalysis is, in many cases, a prerequisite for the cost-effective production of chemicals by biotechnological means. Synthetic metal catalysts for bioorthogonal reactions can be inactivated within cells due to abundant thiol derivatives. Here, a cell surface display-based whole cell biohybrid catalyst system (termed ArMt bugs) is reported as a generally applicable platform to unify cost-effective whole cell catalysis with biohybrid catalysis. An inactivated esterase autotransporter is employed to display the nitrobindin protein scaffold with a Rh catalyst on the E. coli surface. Stereoselective polymerization of phenylacetylene yielded a high turnover number (TON) (39 × 106 cell–1) for the ArMt bugs conversion platform.

Dömötör, O.; Enyedy, É.A.

J. Biol. Inorg. Chem. 2019, 24, 703-719, 10.1007/s00775-019-01683-0

Various half-sandwich ruthenium(II) arene complexes and rhodium(III) arene complexes have been intensively investigated due to their prominent anticancer activity. The interaction of the organometallic complexes of Ru(η6-p-cymene) and Rh(η5-C5Me5) with human serum albumin (HSA) was studied in detail by a combination of various methods such as ultrafiltration, capillary electrophoresis, 1H NMR spectroscopy, fluorometry and UV–visible spectrophotometry in the presence of 100 mM chloride ions. Binding characteristics of the organometallic ions and their complexes with deferiprone, 2-picolinic acid, maltol, 6-methyl-2-picolinic acid and 2-quinaldic acid were evaluated. Kinetic aspects and reversibility of the albumin binding are also discussed. The effect of low-molecular-mass blood components on the protein binding was studied in addition to the interaction of organorhodium complexes with cell culture medium components. The organometallic ions were found to bind to HSA to a high extent via a coordination bond. Release of the bound metal ions was kinetically hindered and could not be induced by the denaturation of the protein. Binding of the Ru(η6-p-cymene) triaqua cation was much slower (ca. 24 h) compared to the rhodium congener (few min), while their complexes interacted with the protein relatively fast (1–2 h). The studied complexes were bound to HSA coordinatively. The highly stable and kinetically inert 2-picolinate Ru(η6-p-cymene) complex bound in an associative manner preserving its original entity, while lower stability complexes decomposed partly or completely upon binding to HSA. Fast, non-specific and high-affinity binding of the complexes on HSA highlights their coordinative interaction with various types of proteins possibly decreasing effective drug concentration.

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.

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.

Whitesides, G.M.

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

n/a

Novič, M.

Mol. Divers. 2007, 11, 141-152, 10.1007/s11030-008-9068-x

The counter propagation artificial neural networks (CP-ANNs) were used to develop a quantitative structure-selectivity relationship (QSSR) for a set of artificial metalloenzymes. The artificial metalloenzymes consist of biotinylated rhodium-diphosphine complexes incorporated in streptavidin mutants acting as host protein. Such hybrid catalysts have been shown to be good enantioselective hydrogenation catalysts for acetamidoacrylic acid. The descriptor-based models were constructed to predict enantiomeric excess (%ee) on the basis of the catalyst structures originating from docking simulations. 3D molecular descriptors for the docked ligands structures were computed. The relative arrangement of guest and host molecules was coded using distance descriptors (Rh-Cα interatomic distances); the diversity of the mutant proteins at the position S112 was coded with molecular descriptors for the sequence of three neighboring amino acids (T111-S112X-G113). The selection of testing samples for the external model validation was based on the Kohonen mapping. The final model trained by two thirds of the entire dataset was characterized by satisfactory statistical parameters for the external test set (R = 0.953 and RMS = 16.8 %ee). The proposed procedure of docking-based descriptor generation thus appears as a promising alternative to the full characterization of the complex structure by experimental or computational methods.

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.

Hayashi, T; Onoda, A.

ChemBioChem 2021, 22, 679-685, 10.1002/cbic.202000681

Directed evolution of Cp*RhIII-linked nitrobindin (NB), a biohybrid catalyst, was performed based on an in vitro screening approach. A key aspect of this effort was the establishment of a high-throughput screening (HTS) platform that involves an affinity purification step employing a starch-agarose resin for a maltose binding protein (MBP) tag. The HTS platform enables efficient preparation of the purified MBP-tagged biohybrid catalysts in a 96-well format and eliminates background influence of the host E. coli cells. Three rounds of directed evolution and screening of more than 4000 clones yielded a Cp*RhIII-linked NB(T98H/L100K/K127E) variant with a 4.9-fold enhanced activity for the cycloaddition of acetophenone oximes with alkynes. It is confirmed that this HTS platform for directed evolution provides an efficient strategy for generating highly active biohybrid catalysts incorporating a synthetic metal cofactor.

Reetz, M.T.

Chem. Commun. 2006, 4318, 10.1039/b610461d

The concept of utilizing the methods of directed evolution for tuning the enantioselectivity of synthetic achiral metal–ligand centers anchored to proteins has been implemented experimentally for the first time.

Marino, T.

ACS Catal. 2015, 5, 5397-5409, 10.1021/acscatal.5b00185

Recently, a new artificial carbonic anhydrase enzyme in which the native zinc cation has been replaced with a Rh(I) has been proposed as a new reductase that is able to efficiently catalyze the hydrogenation of olefins. In this paper, we propose the possible use of this modified enzyme in the direct hydrogenation of carbon dioxide. In our theoretical investigation, we have considered different reaction mechanisms such as reductive elimination and σ-bond metathesis. In addition, the release of the formic acid and the restoring of the catalytic cycle have also been studied. Results show that the σ-bond metathesis potential energy surface lies below the reactant species. The rate-determining step is the release of the product with an energy barrier of 12.8 kcal mol–1. On the basis of our results, we conclude that this artificial enzyme can efficiently catalyze the conversion of CO2 to HCOOH by a direct hydrogenation reaction.