Artificial Metalloproteins Containing Co4O4 Cubane Active Sites
J. Am. Chem. Soc. 2018, 140, 2739-2742, 10.1021/jacs.7b13052
Artificial metalloproteins (ArMs) containing Co4O4 cubane active sites were constructed via biotin–streptavidin technology. Stabilized by hydrogen bonds (H-bonds), terminal and cofacial CoIII–OH2 moieties are observed crystallographically in a series of immobilized cubane sites. Solution electrochemistry provided correlations of oxidation potential and pH. For variants containing Ser and Phe adjacent to the metallocofactor, 1e–/1H+ chemistry predominates until pH 8, above which the oxidation becomes pH-independent. Installation of Tyr proximal to the Co4O4 active site provided a single H-bond to one of a set of cofacial CoIII–OH2 groups. With this variant, multi-e–/multi-H+ chemistry is observed, along with a change in mechanism at pH 9.5 that is consistent with Tyr deprotonation. With structural similarities to both the oxygen-evolving complex of photosystem II (H-bonded Tyr) and to thin film water oxidation catalysts (Co4O4 core), these findings bridge synthetic and biological systems for water oxidation, highlighting the importance of secondary sphere interactions in mediating multi-e–/multi-H+ reactivity.
Metal: CoHost protein: Streptavidin (Sav)Reaction: Electron/Proton transferMax TON: ---ee: ---PDB: 6AUCNotes: Co-complex in Sav WT
Metal: CoHost protein: Streptavidin (Sav)Reaction: Electron/Proton transferMax TON: ---ee: ---PDB: 6AUENotes: Co-complex in Sav S112Y
Asymmetric δ-Lactam Synthesis with a Monomeric Streptavidin Artificial Metalloenzyme
J. Am. Chem. Soc. 2019, 141, 4815-4819, 10.1021/jacs.9b01596
Reliable design of artificial metalloenzymes (ArMs) to access transformations not observed in nature remains a long-standing and important challenge. We report that a monomeric streptavidin (mSav) Rh(III) ArM permits asymmetric synthesis of α,β-unsaturated-δ-lactams via a tandem C–H activation and [4+2] annulation reaction. These products are readily derivatized to enantioenriched piperidines, the most common N-heterocycle found in FDA approved pharmaceuticals. Desired δ-lactams are achieved in yields as high as 99% and enantiomeric excess of 97% under aqueous conditions at room temperature. Embedding a Rh cyclopentadienyl (Cp*) catalyst in the active site of mSav results in improved stereocontrol and a 7-fold enhancement in reactivity relative to the isolated biotinylated Rh(III) cofactor. In addition, mSav-Rh outperforms its well-established tetrameric forms, displaying 11–33 times more reactivity.
Host protein: Streptavidin (monmeric)Reaction: Lactam synthesisMax TON: 33ee: 97PDB: ---Notes: ---
Diruthenium Diacetate-Catalyzed Aerobic Oxidation of Hydroxylamines and Improved Chemoselectivity by Immobilization to Lysozyme
ChemCatChem 2017, 9, 4225-4230, 10.1002/cctc.201701083
A new green method for the preparation of nitrones through the aerobic oxidation of the corresponding N,N‐disubstituted hydroxylamines has been developed upon exploring the catalytic activity of a diruthenium catalyst, that is, [Ru2(OAc)4Cl]), in aqueous or alcoholic solution under mild reaction conditions (0.1 to 1 mol % catalyst, air, 50 °C) and reasonable reaction times. Notably, the catalytic activity of the dimetallic centre is retained after its binding to the small protein lysozyme. Interestingly, this new artificial metalloenzyme conferred complete chemoselectivity to the oxidation of cyclic hydroxylamines, in contrast to the diruthenium catalyst.
Metal: RuLigand type: Amino acid; OAcHost protein: LysozymeAnchoring strategy: DativeOptimization: ChemicalReaction: Oxidation of hydroxylaminesMax TON: 1000ee: ---PDB: ---Notes: ---
Evolving Artificial Metalloenzymes via Random Mutagenesis
Nat. Chem. 2018, 10, 318-324, 10.1038/nchem.2927
Random mutagenesis has the potential to optimize the efficiency and selectivity of protein catalysts without requiring detailed knowledge of protein structure; however, introducing synthetic metal cofactors complicates the expression and screening of enzyme libraries, and activity arising from free cofactor must be eliminated. Here we report an efficient platform to create and screen libraries of artificial metalloenzymes (ArMs) via random mutagenesis, which we use to evolve highly selective dirhodium cyclopropanases. Error-prone PCR and combinatorial codon mutagenesis enabled multiplexed analysis of random mutations, including at sites distal to the putative ArM active site that are difficult to identify using targeted mutagenesis approaches. Variants that exhibited significantly improved selectivity for each of the cyclopropane product enantiomers were identified, and higher activity than previously reported ArM cyclopropanases obtained via targeted mutagenesis was also observed. This improved selectivity carried over to other dirhodium-catalysed transformations, including N–H, S–H and Si–H insertion, demonstrating that ArMs evolved for one reaction can serve as starting points to evolve catalysts for others.
Reaction: CyclopropanationMax TON: 66ee: 94Notes: Mutagenesis of the ArM by error-prone PCR
Reaction: N-H InsertionMax TON: 73ee: 40Notes: Mutagenesis of the ArM by error-prone PCR
Reaction: S-H insertionMax TON: 64ee: 32Notes: Mutagenesis of the ArM by error-prone PCR
Reaction: Si-H insertionMax TON: 35ee: 64Notes: Mutagenesis of the ArM by error-prone PCR