Fujieda, N.; Itoh, S.

Chem. - Asian J. 2012, 7, 1203-1207, 10.1002/asia.201101014

Teaching metalloenzymes new tricks: An artificial type III dicopper oxidase has been developed using a hydrolytic enzyme, metallo‐β‐lactamase, as a metal‐binding platform. The triple mutant D88G/S185H/P224G redesigned by computer‐assisted structural analysis showed spectroscopic features similar to those of type III copper proteins and exhibited a high catalytic activity in the oxidation of catechols under aerobic conditions.

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.

Harada, A.; Yamaguchi, H.

Sci. Rep. 2019, 9, 10.1038/s41598-019-49844-0

Design and engineering of protein scaffolds are crucial to create artificial metalloenzymes. Herein we report the first example of C-C bond formation catalyzed by artificial metalloenzymes, which consist of monoclonal antibodies (mAbs) and C2 symmetric metal catalysts. Prepared as a tailored protein scaffold for a binaphthyl derivative (BN), mAbs bind metal catalysts bearing a 1,1?-bi-isoquinoline (BIQ) ligand to yield artificial metalloenzymes. These artificial metalloenzymes catalyze the Friedel-Crafts alkylation reaction. In the presence of mAb R44E1, the reaction proceeds with 88% ee. The reaction catalyzed by Cu-catalyst incorporated into the binding site of mAb R44E1 is found to show excellent enantioselectivity with 99% ee. The protein environment also enables the use of BIQ-based catalysts as asymmetric catalysts for the first time.

Fujieda, N.; Itoh, S.

J. Am. Chem. Soc. 2017, 139, 5149-5155, 10.1021/jacs.7b00675

Thermally stable TM1459 cupin superfamily protein from Thermotoga maritima was repurposed as an osmium (Os) peroxygenase by metal-substitution strategy employing the metal-binding promiscuity. This novel artificial metalloenzyme bears a datively bound Os ion supported by the 4-histidine motif. The well-defined Os center is responsible for not only the catalytic activity but also the thermodynamic stability of the protein folding, leading to the robust biocatalyst (Tm ≈ 120 °C). The spectroscopic analysis and atomic resolution X-ray crystal structures of Os-bound TM1459 revealed two types of donor sets to Os center with octahedral coordination geometry. One includes trans-dioxide, OH, and mer-three histidine imidazoles (O3N3 donor set), whereas another one has four histidine imidazoles plus OH and water molecule in a cis position (O2N4 donor set). The Os-bound TM1459 having the latter donor set (O2N4 donor set) was evaluated as a peroxygenase, which was able to catalyze cis-dihydroxylation of several alkenes efficiently. With the low catalyst loading (0.01% mol), up to 9100 turnover number was achieved for the dihydroxylation of 2-methoxy-6-vinyl-naphthalene (50 mM) using an equivalent of H2O2 as oxidant at 70 °C for 12 h. When octene isomers were dihydroxylated in a preparative scale for 5 h (2% mol cat.), the terminal alkene octene isomers was converted to the corresponding diols in a higher yield as compared with the internal alkenes. The result indicates that the protein scaffold can control the regioselectivity by the steric hindrance. This protein scaffold enhances the efficiency of the reaction by suppressing disproportionation of H2O2 on Os reaction center. Moreover, upon a simple site-directed mutagenesis, the catalytic activity was enhanced by about 3-fold, indicating that Os-TM1459 is evolvable nascent osmium peroxygenase.

Fujieda, N.; Itoh, S.

Angew. Chem. 2020, 132, 7791-7794, 10.1002/ange.202000129

Cupin superfamily proteins (TM1459) work as a macromolecular ligand framework with a double-stranded β-barrel structure ligating to a Cu ion through histidine side chains. Variegating the first coordination sphere of TM1459 revealed that H52A and H54A/H58A mutants effectively catalyzed the diastereo- and enantioselective Michael addition reaction of nitroalkanes to an α,β-unsaturated ketone. Moreover, calculated substrate docking signified C106N and F104W single-point mutations, which inverted the diastereoselectivity of H52A and further improved the stereoselectivity of H54A/H58A, respectively.

Fujieda, N.; Ward, T.R.

Chem. Sci. 2015, 6, 4060-4065, 10.1039/c5sc01065a

Adding a metal cofactor to a protein bearing a latent metal binding site endows the macromolecule with nascent catalytic activity.

Harada, A.

Chem. Lett. 2008, 37, 1184-1189, 10.1246/cl.2008.1184

Monoclonal antibodies have been prepared against water-soluble porphyrins, viologen derivatives, and transition-metal complexes, respectively. These monoclonal antibodies were utilized to devise biosensing and catalytic systems.

Hyster, T.K.; Ward, T.R.

Angew. Chem. Int. Ed. 2016, 55, 7344-7357, 10.1002/anie.201508816

Artificial metalloenzymes have received increasing attention over the last decade as a possible solution to unaddressed challenges in synthetic organic chemistry. Whereas traditional transition‐metal catalysts typically only take advantage of the first coordination sphere to control reactivity and selectivity, artificial metalloenzymes can modulate both the first and second coordination spheres. This difference can manifest itself in reactivity profiles that can be truly unique to artificial metalloenzymes. This Review summarizes attempts to modulate the second coordination sphere of artificial metalloenzymes by using genetic modifications of the protein sequence. In doing so, successful attempts and creative solutions to address the challenges encountered are highlighted.

Harada, A.

Chem. - Eur. J. 2004, 10, 6179-6186, 10.1002/chem.200305692

Peroxidase activity of a complex of water‐soluble cationic metalloporphyrin with anti‐cationic porphyrin antibody is reported. Antibody 12E11G, which was prepared by immunization with a conjugate of 5‐(4‐carboxyphenyl)‐10,15,20‐tris(4‐methylpyridyl)porphine iodide (3MPy1C), bound to tetramethylpyridylporphyrin iron complex (FeIII–TMPyP) with the dissociation constant of 2.6×10−7 M. The complex of antibody 12E11G with FeIII–TMPyP catalyzed oxidation of pyrogallol, catechol, and guaiacol. A Lineweaver–Burk plot for the oxidation of pyrogallol catalyzed by the FeIII–TMPyP–antibody complex showed Km=8.6 mM and kcat=680 min−1. Under the same conditions, Km and kcat for horseradish peroxidase (HRP) were 0.8 mM and 1750 min−1, respectively. Although the binding interaction of the antibody to the substrates was one order lower than that of native HRP, the peroxidase activity of this system was in the same order of magnitude as that of HRP.

Harada, A.

Inorg. Chem. 1997, 36, 6099-6102, 10.1021/ic9610849

Antibody 03-1, which was prepared by immunization with meso-tetrakis(4-carboxyphenyl)porphyrin (TCPP) conjugate, has been found to bind strongly to Mn(III)−TCPP and Fe(III)−TCPP complexes with dissociation constants of 4.1 × 10-7 and 1.5 × 10-7 M, respectively, although other monoclonal antibodies raised against TCPP did not bind to these TCPP−metal complexes. The complexes of antibody 03-1 with Mn(III)−TCPP and Fe(III)−TCPP were found to catalyze oxidation of pyrogallol selectively. A Lineweaver-Burk plot for the oxidation of pyrogallol by the antibody−Fe−TCPP complex showed Km = 4.0 mM and kcat = 50 min-1. Studies on the effect of the molar ratio of the antibody to metalloporphyrin on the catalytic activity showed that a 1:1 complex was the most effective for the reaction. The effect of salt (NaCl) on the reaction showed that electrostatic interaction between the antibody and the metalloporphyrin was important for the reaction. The antibody−metalloporphyrin complexes are stable enough to show catalytic activity in the presence of an excess amount of H2O2.