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.

Tiller, J.C.

ChemCatChem 2016, 8, 593-599, 10.1002/cctc.201501083

The Sharpless dihydroxylation of styrene with the artificial metalloenzyme osmate‐laccase‐poly(2‐methyloxazoline) was investigated to find reaction conditions that allow this unique catalyst to reveal its full potential. After changing the co‐oxidizing agent to tert‐butyl hydroperoxide and optimizing the osmate/enzyme ratio, the turnover frequency and the turnover number could be increased by an order of magnitude, showing that the catalyst can compete with classical organometallic catalysts. Varying the metal in the active center showed that osmate is by far the most active catalytic center, but the reaction can also be realized with permanganate and iron(II) salts.

Ward, T.R.

Angew. Chem. Int. Ed. 2011, 50, 10863-10866, 10.1002/anie.201103632

Taking control: Selective catalysts for olefin dihydroxylation have been generated by the combination of apo‐streptavidin and OsO4. Site‐directed mutagenesis allows improvement of enantioselectivity and even inversion of enantiopreference in certain cases. Notably allyl phenyl sulfide and cis‐β‐methylstyrene were converted with unprecedented enantiomeric excess.

Tiller, J.C.

ChemBioChem 2015, 16, 83-90, 10.1002/cbic.201402339

Count Os in: We report organosoluble artificial metalloenzymes, generated from poly(2‐methyl‐oxazoline) enzyme conjugates and osmate as a promising new catalytic system for the dihydroxylation of alkenes in organic media.

Kokubo, T.; Okano, M.

J. Chem. Soc., Chem. Commun. 1983, 0, 769-770, 10.1039/C39830000769

The 1:1 complex between an osmate ester and bovine serum albumin was found to be effective as an enantioselective catalyst in the cis-hydroxylation of alkenes, affording diols in up to 68% e.e. and turnover of the catalyst with t-butyl hydroperoxide.