Ward, T.R.

J. Am. Chem. Soc. 2008, 130, 8085-8088, 10.1021/ja8017219

Nature’s catalysts are specifically evolved to carry out efficient and selective reactions. Recent developments in biotechnology have allowed the rapid optimization of existing enzymes for enantioselective processes. However, the ex nihilo creation of catalytic activity from a noncatalytic protein scaffold remains very challenging. Herein, we describe the creation of an artificial enzyme upon incorporation of a vanadyl ion into the biotin-binding pocket of streptavidin, a protein devoid of catalytic activity. The resulting artificial metalloenzyme catalyzes the enantioselective oxidation of prochiral sulfides with good enantioselectivities both for dialkyl and alkyl-aryl substrates (up to 93% enantiomeric excess). Electron paragmagnetic resonance spectroscopy, chemical modification, and mutagenesis studies suggest that the vanadyl ion is located within the biotin-binding pocket and interacts only via second coordination sphere contacts with streptavidin.

Ward, T.R.

Coord. Chem. Rev. 2008, 252, 751-766, 10.1016/j.ccr.2007.09.016

The fusion of homogeneous and enzymatic catalysis has recently drawn attention due to reported novel activities and high selectivities. The incorporation of metal-catalysts into proteins combines the advantages of both catalytic strategies. Herein we summarize recent approaches of artificial metalloenzymes applied to catalysis. The discussion includes different strategies of anchoring and screening for improved selectivity.

Ward, T.R.

Synlett 2009, 2009, 3225-3236, 10.1055/s-0029-1218305

By combining homogeneous with enzymatic catalysis, artificial metalloenzymes offer new perspectives for conferring unnatural activities to biomolecules. The article reassembles the important advances in the field of these hybrid catalysts and summarizes the contributions of our group to this continuously growing field of research.

Ward, T.R.

Angew. Chem. Int. Ed. 2008, 47, 701-705, 10.1002/anie.200703159

Palladium in the active site: The incorporation of a biotinylated palladium diphosphine within streptavidin yielded an artificial metalloenzyme for the title reaction (see scheme). Chemogenetic optimization of the catalyst by the introduction of a spacer (red star) between biotin (green triangle) and palladium and saturation mutagenesis at position S112X afforded both R‐ and S‐selective artificial asymmetric allylic alkylases.

Ward, T.R.

Chimia 2008, 62, 956-961, 10.2533/chimia.2008.956

Artificial metalloenzymes, based on the incorporation of a biotinylated catalytically active organometallic moiety within streptavidin, offer an attractive alternative to homogeneous, heterogeneous and enzymatic catalysis. In this account, we outline our recent results and implications in the developments of such artificial metalloenzymes for various asymmetric transformations, including hydrogenation, transfer hydrogenation, allylic alkylation and sulfoxidation.

Ward, T.R.

Top. Organomet. Chem. 2009, 10.1007/3418_2008_3

Artificial metalloenzymes can be created by incorporating an active metal catalyst precursor in a macromolecular host. When considering such artificial metalloenzymes, the first point to address is how to localize the active metal moiety within the protein scaffold. Although a covalent anchoring strategy may seem most attractive at first, supramolecular anchoring strategy has proven most successful thus far. In this context and inspired by Whitesides’ seminal paper, we have exploited the biotin–avidin technology to anchor a biotinylated active metal catalyst precursor within either avidin or streptavidin. A combined chemical and genetic strategy allows a rapid (chemogenetic) optimization of both the activity and the selectivity of the resulting artificial metalloenzymes. The chiral environment, provided by second coordination sphere interactions between the metal and the host protein, can be varied by introduction of a spacer between the biotin anchor and the metal moiety or by variation of the ligand scaffold. Alternatively, mutagenesis of the host protein allows a fine tuning of the activity and the selectivity. With this protocol, we have been able to produce artificial metalloenzymes based on the biotin–avidin technology for the enantioselective hydrogenation of N-protected dehydroaminoacids, the transfer hydrogenation of prochiral ketones as well as the allylic alkylation of symmetric substrates. In all cases selectivities >90% were achieved. Most recently, guided by an X-ray structure of an artificial metalloenzyme, we have extended the chemogenetic optimization to a designed evolution scheme. Designed evolution combines rational design with combinatorial screening. In this chapter, we emphasize the similarities and the differences between artificial metalloenzymes and their homogeneous or enzymatic counterparts.

Ward, T.R.

Chem. Commun. 2008, 4239, 10.1039/b806652c

Artificial metalloenzymes, based on the incorporation of a catalytically active organometallic moiety within a host protein, lie at the interface between organometallic and enzymatic catalysis. In terms of activity, reaction repertoire, substrate range and operating conditions, they take advantage of the versatility of the organometallic chemistry. In contrast, the enantioselectivity is determined by the biomolecular scaffold, which provides a well defined second coordination sphere to the organometallic moiety, reminiscent of enzymes. The attractive feature of such systems is their optimization potential, which combines chemical and genetic methods (i.e. chemogenetic) to screen diversity space. This feature article describes the implementation of such an optimization protocol for artificial transfer hydrogenases, for which we have the most detailed understanding.

Ward, T.R.

Org. Biomol. Chem. 2007, 5, 1835, 10.1039/b702068f

Artificial metalloenzymes based on biotin–streptavidin technology, a “fusion” of chemistry and biology, illustrate how asymmetric catalysts can be improved and evolved using chemogenetic approaches.

Ward, T.R.

J. Organomet. Chem. 2009, 694, 930-936, 10.1016/j.jorganchem.2008.11.023

Incorporation of achiral biotinylated manganese-salen complexes into streptavidin yields artificial metalloenzymes for aqueous sulfoxidation using hydrogen peroxide. Four biotinylated salen ligands were synthesized and their manganese complexes were tested in combination with several streptavidin mutants, yielding moderate conversions (up to 56%) and low enantioselectivities (maximum of 13% ee) for the sulfoxidation of thioanisole.

Ward, T.R.

Adv. Synth. Catal. 2007, 349, 1923-1930, 10.1002/adsc.200700022

We report on our efforts to create efficient artificial metalloenzymes for the enantioselective hydrogenation of N‐protected dehydroamino acids using either avidin or streptavidin as host proteins. Introduction of chiral amino acid spacers – phenylalanine or proline – between the biotin anchor and the flexible aminodiphosphine moiety 1, combined with saturation mutagenesis at position S112X of streptavidin, affords second generation artificial hydrogenases displaying improved organic solvent tolerance, reaction rates (3‐fold) and (S)‐selectivities (up to 95 % ee for N‐acetamidoalanine and N‐acetamidophenylalanine). It is shown that these artificial metalloenzymes follow Michaelis–Menten kinetics with an increased affinity for the substrate and a higher kcat than the protein‐free catalyst (compare kcat 3.06 min−1 and KM 7.38 mM for [Rh(COD)Biot‐1]+ with kcat 12.30 min−1 and KM 4.36 mM for [Rh(COD)Biot‐(R)‐Pro‐1]+ ⊂ WT Sav). Finally, we present a straightforward protocol using Biotin‐Sepharose to immobilize artificial metalloenzymes (>92 % ee for N‐acetamidoalanine and N‐acetamidophenylalanine using [Rh(COD)Biot‐(R)‐Pro‐1]+ ⊂ Sav S112W).

Ward, T.R.

C. R. Chim. 2007, 10, 678-683, 10.1016/j.crci.2007.02.020

We report on our efforts to create efficient artificial metalloenzymes for the enantioselective hydrogenation of N-protected dehydroamino acids using streptavidin as host protein. Introduction of an (R)-proline spacer between the biotin anchor and the diphosphine moiety affords a versatile ligand Biot-(R)-Pro-1 which displays good (S)-selectivities in the presence of streptavidin (91% ee). The resulting artificial metalloenzyme [Rh(Biot-(R)-Pro-1)(COD)]+ ⊂ WT-Sav displays increased stability against organic solvents.

Ward, T.R.

Angew. Chem. Int. Ed. 2008, 47, 1400-1404, 10.1002/anie.200704865

A structure is worth a thousand words: Guided by the X‐ray structure of an S‐selective artificial transfer hydrogenase, designed evolution was used to optimize the selectivity of hybrid catalysts. Fine‐tuning of the second coordination sphere of the ruthenium center (see picture, orange sphere) by introduction of two point mutations allowed the identification of selective artificial transfer hydrogenases for the reduction of dialkyl ketones.