2 publications
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Library Design and Screening Protocol for Artificial Metalloenzymes Based on the Biotin-Streptavidin Technology
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Nat. Protoc. 2016, 11, 835-852, 10.1038/nprot.2016.019
Artificial metalloenzymes (ArMs) based on the incorporation of a biotinylated metal cofactor within streptavidin (Sav) combine attractive features of both homogeneous and enzymatic catalysts. To speed up their optimization, we present a streamlined protocol for the design, expression, partial purification and screening of Sav libraries. Twenty-eight positions have been subjected to mutagenesis to yield 335 Sav isoforms, which can be expressed in 24-deep-well plates using autoinduction medium. The resulting cell-free extracts (CFEs) typically contain >1 mg of soluble Sav. Two straightforward alternatives are presented, which allow the screening of ArMs using CFEs containing Sav. To produce an artificial transfer hydrogenase, Sav is coupled to a biotinylated three-legged iridium pianostool complex Cp*Ir(Biot-p-L)Cl (the cofactor). To screen Sav variants for this application, you would determine the number of free binding sites, treat them with diamide, incubate them with the cofactor and then perform the reaction with your test compound (the example used in this protocol is 1-phenyl-3,4-dihydroisoquinoline). This process takes 20 d. If you want to perform metathesis reactions, Sav is coupled to a biotinylated second-generation Grubbs-Hoveyda catalyst. In this application, it is best to first immobilize Sav on Sepharose-iminobiotin beads and then perform washing steps. Elution from the beads is achieved in an acidic reaction buffer before incubation with the cofactor. Catalysis using your test compound (in this protocol, 2-(4-(N,N-diallylsulfamoyl)phenyl)-N,N,N-trimethylethan-1-aminium iodide) is performed using the formed metalloenzyme. Screening using this approach takes 19 d.
Metal: IrHost protein: Streptavidin (Sav)Anchoring strategy: SupramolecularOptimization: Chemical & geneticNotes: Purified streptavidin (mutant K121A)
Metal: IrHost protein: Streptavidin (Sav)Anchoring strategy: SupramolecularOptimization: Chemical & geneticNotes: Cell free extract (mutant Sav K121A) treated with diamide
Metal: RuLigand type: N-heterocyclic carbeneHost protein: Streptavidin (Sav)Anchoring strategy: SupramolecularOptimization: Chemical & geneticNotes: Purified streptavidin (mutant K121A)
Metal: RuLigand type: N-heterocyclic carbeneHost protein: Streptavidin (Sav)Anchoring strategy: SupramolecularOptimization: Chemical & geneticNotes: Cell free extract (mutant Sav K121A immobilised on iminobiotin-sepharose beads)
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Manganese Terpyridine Artificial Metalloenzymes for Benzylic Oxygenation and Olefin Epoxidation
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Tetrahedron 2014, 70, 4245-4249, 10.1016/j.tet.2014.03.008
New catalysts for non-directed hydrocarbon functionalization have great potential in organic synthesis. We hypothesized that incorporating a Mn-terpyridine cofactor into a protein scaffold would lead to artificial metalloenzymes (ArMs) in which the selectivity of the Mn cofactor could be controlled by the protein scaffold. We designed and synthesized a maleimide-substituted Mn-terpyridine cofactor and demonstrated that this cofactor could be incorporated into two different scaffold proteins to generate the desired ArMs. The structure and reactivity of one of these ArMs was explored, and the broad oxygenation capability of the Mn-terpyridine catalyst was maintained, providing a robust platform for optimization of ArMs for selective hydrocarbon functionalization.
Metal: MnLigand type: Poly-pyridineHost protein: Nitrobindin (Nb)Anchoring strategy: CovalentOptimization: ChemicalNotes: ---
Metal: MnLigand type: Poly-pyridineHost protein: Nitrobindin (Nb)Anchoring strategy: CovalentOptimization: ChemicalNotes: ---