Okuda, J.

ACS Catal. 2015, 5, 7519-7522, 10.1021/acscatal.5b01792

A series of Grubbs–Hoveyda type catalyst precursors for olefin metathesis containing a maleimide moiety in the backbone of the NHC ligand was covalently incorporated in the cavity of the β-barrel protein nitrobindin. By using two protein mutants with different cavity sizes and choosing the suitable spacer length, an artificial metalloenzyme for olefin metathesis reactions in water in the absence of any organic cosolvents was obtained. High efficiencies reaching TON > 9000 in the ROMP of a water-soluble 7-oxanorbornene derivative and TON > 100 in ring-closing metathesis (RCM) of 4,4-bis(hydroxymethyl)-1,6-heptadiene in water under relatively mild conditions (pH 6, T = 25–40 °C) were observed.

Okuda, J.; Schwaneberg, U.

Chem. - Eur. J. 2013, 19, 13865-13871, 10.1002/chem.201301515

A β‐barrel protein hybrid catalyst was prepared by covalently anchoring a Grubbs–Hoveyda type olefin metathesis catalyst at a single accessible cysteine amino acid in the barrel interior of a variant of β‐barrel transmembrane protein ferric hydroxamate uptake protein component A (FhuA). Activity of this hybrid catalyst type was demonstrated by ring‐opening metathesis polymerization of a 7‐oxanorbornene derivative in aqueous solution.

Hilvert, D.; Ward, T.R.

Chem. Commun. 2011, 47, 12068, 10.1039/c1cc15005g

A Grubbs–Hoveyda type olefin metathesis catalyst, equipped with an electrophilic bromoacetamide group, was used to modify a cysteine-containing variant of a small heat shock protein from Methanocaldococcus jannaschii. The resulting artificial metalloenzyme was found to be active under acidic conditions in a benchmark ring closing metathesis reaction.

Ward, T.R.

Chem. Sci. 2016, 7, 673-677, 10.1039/c5sc03116h

Introduction of a biotinylated monophosphine palladium complex within streptavidin affords an enantioselective artificial Suzukiase. Site-directed mutagenesis allowed the optimization of the activity and the enantioselectivity of this artificial metalloenzyme. A variety of atropisomeric biaryls were produced in good yields and up to 90% ee.

Ward, T.R.

Chem. Commun. 2011, 47, 12065, 10.1039/c1cc15004a

Incorporation of a biotinylated Hoveyda-Grubbs catalyst within (strept)avidin affords artificial metalloenzymes for the ring-closing metathesis of N-tosyl diallylamine in aqueous solution. Optimization of the performance can be achieved either by chemical or genetic means.

Ward, T.R.

Org. Biomol. Chem. 2015, 13, 5652-5655, 10.1039/c5ob00428d

We report an efficient artificial metathesase which combines an arylsulfonamide anchor within the protein scaffold human carbonic anhydrase II.

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.

Chem. Commun. 2012, 48, 1662, 10.1039/c2cc16898g

An L-phenylalanyl chloromethylketone-based inhibitor equipped with a Hoveyda–Grubbs catalyst moiety was regioselectively incorporated into the cleft of α-chymotrypsin through the intrinsic inhibition mechanism of the protein to construct an artificial organometallic protein.

Panke, S.; Ward, T.R.

Nature 2016, 537, 661-665, 10.1038/nature19114

The field of biocatalysis has advanced from harnessing natural enzymes to using directed evolution to obtain new biocatalysts with tailor-made functions1. Several tools have recently been developed to expand the natural enzymatic repertoire with abiotic reactions2,3. For example, artificial metalloenzymes, which combine the versatile reaction scope of transition metals with the beneficial catalytic features of enzymes, offer an attractive means to engineer new reactions. Three complementary strategies exist3: repurposing natural metalloenzymes for abiotic transformations2,4; in silico metalloenzyme (re-)design5,6,7; and incorporation of abiotic cofactors into proteins8,9,10,11. The third strategy offers the opportunity to design a wide variety of artificial metalloenzymes for non-natural reactions. However, many metal cofactors are inhibited by cellular components and therefore require purification of the scaffold protein12,13,14,15. This limits the throughput of genetic optimization schemes applied to artificial metalloenzymes and their applicability in vivo to expand natural metabolism. Here we report the compartmentalization and in vivo evolution of an artificial metalloenzyme for olefin metathesis, which represents an archetypal organometallic reaction16,17,18,19,20,21,22 without equivalent in nature. Building on previous work6 on an artificial metallohydrolase, we exploit the periplasm of Escherichia coli as a reaction compartment for the ‘metathase’ because it offers an auspicious environment for artificial metalloenzymes, mainly owing to low concentrations of inhibitors such as glutathione, which has recently been identified as a major inhibitor15. This strategy facilitated the assembly of a functional metathase in vivo and its directed evolution with substantially increased throughput compared to conventional approaches that rely on purified protein variants. The evolved metathase compares favourably with commercial catalysts, shows activity for different metathesis substrates and can be further evolved in different directions by adjusting the workflow. Our results represent the systematic implementation and evolution of an artificial metalloenzyme that catalyses an abiotic reaction in vivo, with potential applications in, for example, non-natural metabolism.

Okuda, J.

Chem. - Asian J. 2015, 10, 177-182, 10.1002/asia.201403005

A biohybrid ring‐opening olefin metathesis polymerization catalyst based on the reengineered β‐barrel protein FhuA ΔCVFtev was chemically modified with respect to the covalently anchored Grubbs–Hoveyda type catalyst. Shortening of the spacer (1,3‐propanediyl to methylene) between the N‐heterocyclic carbene ligand and the cysteine site 545 increased the ROMP activity toward a water‐soluble 7‐oxanorbornene derivative. The cis/trans ratio of the double bond in the polymer was influenced by the hybrid catalyst.

Klein Gebbink, R.J.M.

Chem. Commun. 2015, 51, 6792-6795, 10.1039/c4cc09700a

A Rh(NHC) phosphonate complex reacts with the lipases cutinase and Candida antarctica lipase B resulting in the first (soluble) artificial metalloenzymes formed by covalent active site-directed hybridization. When compared to unsupported complexes, these new robust hybrids show enhanced chemoselectivity in the (competitive) hydrogenation of olefins over ketones.

Klein Gebbink, R.J.M.

Chem. - Eur. J. 2015, 21, 15676-15685, 10.1002/chem.201502381

A series of Grubbs‐type catalysts that contain lipase‐inhibiting phosphoester functionalities have been synthesized and reacted with the lipase cutinase, which leads to artificial metalloenzymes for olefin metathesis. The resulting hybrids comprise the organometallic fragment that is covalently bound to the active amino acid residue of the enzyme host in an orthogonal orientation. Differences in reactivity as well as accessibility of the active site by the functionalized inhibitor became evident through variation of the anchoring motif and substituents on the N‐heterocyclic carbene ligand. Such observations led to the design of a hybrid that is active in the ring‐closing metathesis and the cross‐metathesis of N,N‐diallyl‐p‐toluenesulfonamide and allylbenzene, respectively, the latter being the first example of its kind in the field of artificial metalloenzymes.