Reetz, M.T.

Angew. Chem. Int. Ed. 2010, 49, 5151-5155, 10.1002/anie.201002106

Guided by nature: A designed binding site comprising the His/His/Asp motif for CuII complexation has been constructed in a robust protein by site‐specific mutagenesis (see picture). The artificial metalloenzyme catalyzes an enantioselective Diels–Alder reaction.

Artero, V.

ChemPlusChem 2016, 81, 1083-1089, 10.1002/cplu.201600218

The insertion of cobaloxime catalysts in the heme‐binding pocket of heme oxygenase (HO) yields artificial hydrogenases active for H2 evolution in neutral aqueous solutions. These novel biohybrids have been purified and characterized by using UV/visible and EPR spectroscopy. These analyses revealed the presence of two distinct binding conformations, thereby providing the cobaloxime with hydrophobic and hydrophilic environments, respectively. Quantum chemical/molecular mechanical docking calculations found open and closed conformations of the binding pocket owing to mobile amino acid residues. HO‐based biohybrids incorporating a {Co(dmgH)2} (dmgH2=dimethylglyoxime) catalytic center displayed up to threefold increased turnover numbers with respect to the cobaloxime alone or to analogous sperm whale myoglobin adducts. This study thus provides a strong basis for further improvement of such biohybrids, using well‐designed modifications of the second and outer coordination spheres, through site‐directed mutagenesis of the host protein.

Reetz, M.T.

Chem. Rec. 2012, 12, 391-406, 10.1002/tcr.201100043

Numerous enzymes are useful catalysts in synthetic organic chemistry, but they cannot catalyze the myriad transition‐metal‐mediated transformations customary in daily chemical work. For this reason the concept of directed evolution of hybrid catalysts was proposed some time ago. A synthetic ligand/transition‐metal moiety is anchored covalently or non‐covalently to a host protein, thereby generating a single artificial metalloenzyme which can then be optimized by molecular biological methods. In the quest to construct an appropriate experimental platform for asymmetric Diels–Alder reactions amenable to this Darwinian approach to catalysis, specifically those not currently possible using traditional chiral transition‐metal catalysts, two strategies have been developed which are reviewed here. One concerns the supramolecular anchoring of a Cu(II)‐phthalocyanine complex to serum albumins; the other is based on the design of a Cu(II)‐specific binding site in a thermostable protein host (tHisF), leading to 46–98% ee in a model Diels–Alder reaction. This sets the stage for genetic fine‐tuning using the methods of directed evolution.

Artero, V.

Inorg. Chem. 2014, 53, 8071-8082, 10.1021/ic501014c

Cobaloximes are popular H2 evolution molecular catalysts but have so far mainly been studied in nonaqueous conditions. We show here that they are also valuable for the design of artificial hydrogenases for application in neutral aqueous solutions and report on the preparation of two well-defined biohybrid species via the binding of two cobaloxime moieties, {Co(dmgH)2} and {Co(dmgBF2)2} (dmgH2 = dimethylglyoxime), to apo Sperm-whale myoglobin (SwMb). All spectroscopic data confirm that the cobaloxime moieties are inserted within the binding pocket of the SwMb protein and are coordinated to a histidine residue in the axial position of the cobalt complex, resulting in thermodynamically stable complexes. Quantum chemical/molecular mechanical docking calculations indicated a coordination preference for His93 over the other histidine residue (His64) present in the vicinity. Interestingly, the redox activity of the cobalt centers is retained in both biohybrids, which provides them with the catalytic activity for H2 evolution in near-neutral aqueous conditions.

Ueno, T.

Small 2010, 6, 1873-1879, 10.1002/smll.201000772

The synthesis of a robust bio‐nanotube consisting of the β‐helical tubular component proteins of bacteriophage T4 is described. The crystal structure indicates that it has a well‐defined nanoscale length of 10 nm as a result of the head‐to‐head dimerization of β‐helices. Surprisingly, the tube assembly has high thermal stability, high tolerance to organic solvents, and a wide pH‐stability range.

Ueno, T.; Watanabe, Y.

J. Am. Chem. Soc. 2008, 130, 10512-10514, 10.1021/ja802463a

We report the preparation of organometallic Pd(allyl) dinuclear complexes in protein cages of apo-Fr by reactions with [Pd(allyl)Cl]2 (allyl = η3-C3H5). One of the dinuclear complexes is converted to a trinuclear complex by replacing a Pd-coordinated His residue to an Ala residue. These results suggest that multinuclear metal complexes with various coordination structures could be prepared by the deletion or introduction of His, Cys, and Glu at appropriate positions on protein surface.

Reetz, M.T.

Angew. Chem. Int. Ed. 2006, 45, 2416-2419, 10.1002/anie.200504561

Chirality from blood: Serum albumins form strong complexes with CuII–phthalocyanines, leading to protein conjugates. These hybrid catalysts promote enantioselective Diels–Alder reactions, such as that of azachalcones 1 with cyclopentadiene (2) to give products 3 with 85–98 % ee.

Ueno, T.

Chem. Commun. 2011, 47, 170-172, 10.1039/C0CC02221G

Apo-ferritin (apo-Fr) mutants are used as scaffolds to accommodate palladium (allyl) complexes. Various coordination arrangements of the Pd complexes are achieved by adjusting the positions of cysteine and histidine residues on the interior surface of the apo-Fr cage.

Reetz, M.T.

Isr. J. Chem. 2015, 55, 51-60, 10.1002/ijch.201400087

Transition metal catalysis in asymmetric transformations plays a pivotal role in modern synthetic organic chemistry, with these catalysts being tuned by systematic variation of the chiral ligand. More than three decades ago it was recognized that an alternative approach is possible, namely the anchoring of an achiral ligand/metal entity in an appropriate protein host, with formation of an artificial metalloenzyme (hybrid catalyst). However, this procedure delivers a single transition metal catalyst, with high enantioselectivity being a matter of chance. In view of this restriction, we proposed in 2001/2002 the concept of directed evolution of such hybrid catalysts. The most intensively studied system involves biotinylated phosphine/metal entities which are non‐covalently anchored to streptavidin. The present review summarizes progress in this intriguing area of research. It includes the assessment of the requirements of a given Darwinian system to be successful, and offers hints on how to achieve success in future studies.

Reetz, M.T.

Acc. Chem. Res. 2019, 52, 336-344, 10.1021/acs.accounts.8b00582

Transition metal catalysts mediate a wide variety of chemo-, stereo-, and regioselective transformations, and therefore play a pivotal role in modern synthetic organic chemistry. Steric and electronic effects of ligands provide organic chemists with an exceedingly useful tool. More than four decades ago, chemists began to think about a different approach, namely, embedding achiral ligand/metal moieties covalently or noncovalently in protein hosts with formation of artificial metalloenzymes. While structurally fascinating, this approach led in each case only to a single (bio)catalyst, with its selectivity and activity being a matter of chance. In order to solve this fundamental problem, my group proposed in 2000−2002 the idea of directed evolution of artificial metalloenzymes. In earlier studies, we had already demonstrated that directed evolution of enzymes constitutes a viable method for enhancing and inverting the stereoselectivity of enzymes as catalysts inorganic chemistry. We speculated that it should also be possible to manipulate selectivity and activity of artificial metalloenzymes, which would provide organic chemists with a tool for optimizing essentially any transition metal catalyzed reaction type. In order to put this vision into practice, we first turned to the Whitesides system for artificial metalloenzyme formation, comprising a biotinylated diphosphine/Rh moiety, which is anchored noncovalently to avidin or streptavidin. Following intensive optimization, proof of principle was finally demonstrated in 2006, which opened the door to a new research area. This personal Account critically assesses these early studies as well as subsequent efforts from my group focusing on different protein scaffolds, and includes briefly some of the most important current contributions of other groups. Two primary messages emerge: First, since organic chemists continue to be extremely good at designing and implementing man-made transition metal catalysts, often on a large scale, those scientists that are active in the equally intriguing field of directed evolution of artificial metalloenzymes should be moderate when generalizing claims. All factors required for a truly viable catalytic system need to beconsidered, especially activity and ease of upscaling. Second, the most exciting and thus far very rare cases of directed evolution of artificial metalloenzymes are those that focus on selective transformations that are not readily possible using state of the art transition metal catalysts.

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.

Reetz, M.T.

Top. Organomet. Chem. 2009, 10.1007/3418_2008_12

Whereas the directed evolution of stereoselective enzymes provides a useful tool in asymmetric catalysis, generality cannot be claimed because enzymes as catalysts are restricted to a limited set of reaction types. Therefore, a new concept has been proposed, namely directed evolution of hybrid catalysts in which proteins serve as hosts for anchoring ligand/transition metal entities. Accordingly, appropriate genetic mutagenesis methods are applied to the gene of a given protein host, providing after expression a library of mutant proteins. These are purified and a ligand/transition metal anchored site-specifically. Following en masse ee-screening, the best hit is identified, and the corresponding mutant gene is used as a template for another round of mutagenesis, expression, purification, bioconjugation, and screening. This allows for a Darwinian optimization of transition metal catalysts.

Ueno, T.

Chem. Commun. 2011, 47, 2074, 10.1039/C0CC03015E

We have constructed a robust β-helical nanotube from the component proteins of bacteriophage T4 and modified this nanotube with RuII(bpy)3 and ReI(bpy)(CO)3Cl complexes. The photocatalytic system arranged on the tube catalyzes the reduction of CO2 with higher reactivity than that of the mixture of the monomeric forms.

Ueno, T.

Curr. Opin. Chem. Biol. 2018, 43, 68-76, 10.1016/j.cbpa.2017.11.015

Self-assembled proteins have specific functions in biology. With inspiration provided by natural protein systems, several artificial protein assemblies have been constructed via site-specific mutations or metal coordination, which have important applications in catalysis, material and bio-supramolecular chemistry. Similar to natural protein assemblies, protein crystals have been recognized as protein assemblies formed of densely-packed monomeric proteins. Protein crystals can be functionalized with metal ions, metal complexes or nanoparticles via soaking, co-crystallization, creating new metal binding sites by site-specific mutations. The field of protein crystal engineering with metal coordination is relatively new and has gained considerable attention for developing solid biomaterials as well as structural investigations of enzymatic reactions, growth of nanoparticles and catalysis. This review highlights recent and significant research on functionalization of protein crystals with metal coordination and future prospects.

Shoji, O.

RSC Adv. 2019, 9, 18697-18702, 10.1039/c9ra02872b

Iron(III)- and cobalt(III)-9,10,19,20-tetraphenylporphycenes, which possess bulky phenyl groups at the four meso positions of porphycene, were successfully incorporated into the haem acquisition protein HasA secreted by Pseudomonas aeruginosa. Crystal structure analysis revealed that loops surrounding the haem-binding site are highly flexible, remodelling themselves to accommodate bulky metal complexes with significantly different structures from the native haem cofactor.

Ueno, T.

Chem. Commun. 2016, 52, 5463-5466, 10.1039/C6CC00679E

Natural protein-based microcompartments containing multiple enzymes promote cascade reactions within cells. We use the apo-ferritin protein cage to mimic such biocompartments by immobilizing two organometallic Ir and Pd complexes into the single protein cage. Precise locations of the metals and their accumulation mechanism were studied by X-ray crystallography.

Artero, V.

Coord. Chem. Rev. 2014, 270-271, 127-150, 10.1016/j.ccr.2013.12.018

Over the last 15 years, a plethora of research has provided major insights into the structure and function of hydrogenase enzymes. This has led to the important development of chemical models that mimic the inorganic enzymatic co-factors, which in turn has further contributed to the understanding of the specific molecular features of these natural systems that facilitate such large and robust enzyme activities. More recently, efforts have been made to generate guest–host models and artificial hydrogenases, through the incorporation of transition metal-catalysts (guests) into various hosts. This adds a new layer of complexity to hydrogenase-like catalytic systems that allows for better tuning of their activity through manipulation of both the first (the guest) and the second (the host) coordination spheres. Herein we review the aforementioned advances achieved during the last 15 years, in the field of inorganic biomimetic hydrogenase chemistry. After a brief presentation of the enzymes themselves, as well as the early bioinspired catalysts, we review the more recent systems constructed as models for the hydrogenase enzymes, with a specific focus on the various strategies employed for incorporating of synthetic models into supramolecular frameworks and polypeptidic/protein scaffolds, and critically discuss the advantages of such an elaborate approach, with regard to the catalytic performances.

Ueno, T.; Watanabe, Y.

Small 2008, 4, 50-54, 10.1002/smll.200700855

A bionanocup chemical reactor is constructed from a heteroprotein assembly from bacteriophage T4. The preparation of a stable iron(III) porphyrin–bionanocup composite is described. The hydrophobic cup provides a space suitable for the fixation of low‐water‐solubility iron(III) porphyrins. The application of the iron(III) porphyrin–bionanocup composites for the catalysis of sulfoxidation of thioanisoles is demonstrated (see figure).

Ueno, T.; Watanabe, Y.

J. Am. Chem. Soc. 2009, 131, 6958-6960, 10.1021/ja901234j

Polymerization reactions of phenylacetylene derivatives are promoted by rhodium complexes within the discrete space of apo-ferritin in aqueous media. The catalytic reaction provides polymers with restricted molecular weight and a narrow molecular weight distribution. These results suggest that protein nanocages have potential for use as various reaction spaces through immobilization of metal catalysts on the interior surfaces of the protein cages.

Kitagawa, S.; Ueno, T.

Chem. - Asian J. 2014, 9, 1373-1378, 10.1002/asia.201301347

Porous protein crystals, which are protein assemblies in the solid state, have been engineered to form catalytic vessels by the incorporation of organometallic complexes. Ruthenium complexes in cross‐linked porous hen egg white lysozyme (HEWL) crystals catalyzed the enantioselective hydrogen‐transfer reduction of acetophenone derivatives. The crystals accelerated the catalytic reaction and gave different enantiomers based on the crystal form (tetragonal or orthorhombic). This method represents a new approach for the construction of bioinorganic catalysts from protein crystals.

Kitagawa, S.; Ueno, T.

Isr. J. Chem. 2015, 55, 40-50, 10.1002/ijch.201400097

Construction of artificial metalloenzymes based on protein assemblies is a promising strategy for the development of new catalysts, because the three‐dimensional nanostructures of proteins with defined individual sizes can be used as molecular platforms that allow the arrangement of catalytic active centers on their surfaces. Protein needles/tubes/fibers are suitable for supporting various functional molecules, including metal complexes, synthetic molecules, metal nanoparticles, and enzymes with high densities and precise locations. Compared with bulk systems, the protein tube‐ and fiber‐based materials have higher activities for catalytic reactions and electron transfer, as well as enhanced functions when used in electronic devices. The natural and synthetic protein tubes and fibers are constructed by self‐assembly of monomer proteins or peptides. For more precise designs of arrangements of metal complexes, we have developed a new conceptual framework, based on the isolation of a robust needle structure from the cell‐puncturing domains of a bacteriophage. The artificial protein needle shows great promise for use in creating efficient catalytic systems by providing the means to arrange the locations of various metal complexes on the protein surface. In this account, we discuss the recent development of protein needle‐based metalloenzymes, and the future developments we are anticipating in this field.

Ueno, T.

Dalton Trans. 2012, 41, 11424, 10.1039/C2DT31030A

We have succeeded in preparing semi-synthesized proteins bound to Sc3+ ion which can promote an epoxide ring-opening reaction. The Sc3+ binding site was created on the surface of [(gp5βf)3]2 (N. Yokoi et al., Small, 2010, 6, 1873) by introducing a cysteine residue for conjugation of a bpy moiety using a thiol–maleimide coupling reaction. Three cysteine mutants [(gp5βf_X)3]2 (X = G18C, L47C, N51C) were prepared to introduce a bpy in different positions because it had been reported that Sc3+ ion can serve as a Lewis-acid catalyst for an epoxide ring-opening reaction upon binding of epoxide to bpy and two –ROH groups. G18C_bpy with Sc3+ can accelerate the rate of catalysis of the epoxide ring-opening reaction and has the highest rate of conversion among the three mutants. The value is more than 20 times higher than that of the mixtures of [(gp5βf)3]2/2,2′-bipyridine and L-threonine/2,2′-bipyridine. The elevated activity was obtained by the cooperative effect of stabilizing the Sc3+ coordination and accumulation of substrates on the protein surface. Thus, we expect that the semi-synthetic approach can provide insights into new rational design of artificial metalloenzymes.

Reetz, M.T.

Chimia 2002, 56, 721-723, 10.2533/000942902777679920

The first step in applying the recently proposed concept concerning the application of directed evolution to the creation of selective hybrid catalysts is described, specifically the covalent attachment of Mn-salen moieties and of Cu-, Pd-, and Rh-complexes of dipyridine derivatives as well as the implantation of a diphosphine moiety in a protein, future steps being cycles of mutagenesis/screening.

Ueno, T.

Curr. Opin. Chem. Biol. 2015, 25, 88-97, 10.1016/j.cbpa.2014.12.026

Self-assembled protein cages providing nanosized internal spaces which are capable of encapsulating metal ions/complexes, enzymes/proteins have great potential for use as catalytic nanoreactors in efforts to mimic confined cellular environments for synthetic applications. Despite many uses in biomineralization, drug delivery, bio-imaging and so on, applications in catalysis are relatively rare. Because of their restricted size, protein cages are excellent candidates for use as vessels to exert control over reaction kinetics and product selectivity. Virus capsids with larger internal spaces can encapsulate multiple enzymes and can mimic natural enzymatic reactions. The apo-ferritin cage is known to accommodate various metal ions/complexes and suitable for organic transformation reactions in an aqueous medium. This review highlights the importance, prospects and recent significant research on catalytic reactions using the apo-ferritin cage and virus capsids.