A General Method for Artificial Metalloenzyme Formation through Strain‐Promoted Azide–Alkyne Cycloaddition

Abstract

Strain-promoted azide-alkyne cycloaddition (SPAAC) can be used to generate artificial metalloenzymes (ArMs) from scaffold proteins containing a p-azido-L-phenylalanine (Az) residue and catalytically active bicyclononyne-substituted metal complexes. The high efficiency of this reaction allows rapid ArM formation using Az residues within the scaffold protein in the presence of cysteine residues or various reactive components of cellular lysate. In general, cofactor-based ArM formation allows the use of any desired metal complex to build unique inorganic-protein materials. SPAAC covalent linkage further decouples the native function of the scaffold from the installation process since it is not affected by native amino acid residues; as long as an Az residue can be incorporated, an ArM can be generated. We have demonstrated the scope of this method with respect to both the scaffold and cofactor components and established that the dirhodium ArMs generated can catalyze the decomposition of diazo compounds and both Si-H and olefin insertion reactions involving these carbene precursors.

Preparation, Characterization, and Oxygenase Activity of a Photocatalytic Artificial Enzyme

“…[11] This bioconjugation method allows rapid, site-selective incorporation of cofactors into protein scaffold. To explore the possibility of incorporating Acr + -Mes cofactors into proteins, we prepared BCN-substituted Acr + -Mes 5 (Scheme 1).…”

Genetic Optimization of Metalloenzymes: Enhancing Enzymes for Non‐Natural Reactions

“…[61][62][63] In stark contrast, most abiotic metal-containing cofactors are inhibited in the presence of cellular extracts.T his drawback also severely limits the use of transition-metal catalysts for chemical biology applications. [67] Interestingly, dirhodiumtetracarboxylate motifs have been shown to maintain their catalytic function in cell lysates.…”

“…Lewis and co-workers pioneered the covalent linking of metal cofactors to proteins by using strain-promoted azide-alkyne cycloadditions,w here l-4-azidophenylalanine was genetically introduced through AMBER codon suppression (Figure 21 a). [62] Lewis and co-workers found that aR h II dimer can be introduced into the active site of prolyl oligopeptidase from Pyrococcus furiosus by mutation of the non-natural amino acid residue at position 477 (Z477), and expanding the active site by the introduction of alanine at four positions (E104, F146, K199, and D202). [63] Ther esulting protein, containing five mutations,w as able to effect the cyclopropanation of styrene with donor-acceptor diazoesters in modest yield and enantioselectivity.When histidine was introduced in the active site at position 328 (H328), presumably to decrease conformational freedom and to block one rhodium center,the enantioselectivity increased to 47 % ee and the product was formed in modest yield.…”

Coordination complexes and biomolecules: A wise wedding for catalysis upgrade

“…The constantly increasing understanding of protein-substrate interactions should help to move towards a more rational method for proteins optimization. Among the efforts made to facilitate the elaboration of the systems, one can especially consider the improvement of the anchoring methods [106][107][108]. Thanks to the support of computational modelling and directed evolution [40], virtually all of the existing systems can be further optimized.…”