11 publications

11 publications

A General Method for Artificial Metalloenzyme Formationthrough Strain-Promoted Azide–Alkyne Cycloaddition

Lewis, J.C.

ChemBioChem 2014, 15, 223-227, 10.1002/cbic.201300661

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 when 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 because 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.


Metal: Rh
Ligand type: Poly-carboxylic acid
Host protein: tHisF
Anchoring strategy: Covalent
Optimization: ---
Reaction: Cyclopropanation
Max TON: 81
ee: ---
PDB: 1THF
Notes: ---

Metal: Rh
Ligand type: Poly-carboxylic acid
Host protein: tHisF
Anchoring strategy: Covalent
Optimization: ---
Reaction: Si-H insertion
Max TON: 7
ee: ---
PDB: 1THF
Notes: ---

Artificial Metalloenzymes and Metallopeptide Catalysts for Organic Synthesis

Review

Lewis, J.C.

ACS Catal. 2013, 3, 2954-2975, 10.1021/cs400806a

Transition metal catalysts and enzymes possess unique and often complementary properties that have made them important tools for chemical synthesis. The potential practical benefits of catalysts that combine these properties and a desire to understand how the structure and reactivity of metal and peptide components affect each other have driven researchers to create hybrid metal–peptide catalysts since the 1970s. The hybrid catalysts developed to date possess unique compositions of matter at the inorganic/biological interface that often pose significant challenges from design, synthesis, and characterization perspectives. Despite these obstacles, researchers have developed systems in which secondary coordination sphere effects impart selectivity to metal catalysts, accelerate chemical reactions, and are systematically optimized via directed evolution. This perspective outlines fundamental principles, key developments, and future prospects for the design, preparation, and application of peptide- and protein-based hybrid catalysts for organic transformations.


Notes: ---

Artificial Metalloenzymes: Reaction Scope and Optimization Strategies

Review

Lewis, J.C.; Ward, T.R.

Chem. Rev. 2018, 118, 142-231, 10.1021/acs.chemrev.7b00014

The incorporation of a synthetic, catalytically competent metallocofactor into a protein scaffold to generate an artificial metalloenzyme (ArM) has been explored since the late 1970’s. Progress in the ensuing years was limited by the tools available for both organometallic synthesis and protein engineering. Advances in both of these areas, combined with increased appreciation of the potential benefits of combining attractive features of both homogeneous catalysis and enzymatic catalysis, led to a resurgence of interest in ArMs starting in the early 2000’s. Perhaps the most intriguing of potential ArM properties is their ability to endow homogeneous catalysts with a genetic memory. Indeed, incorporating a homogeneous catalyst into a genetically encoded scaffold offers the opportunity to improve ArM performance by directed evolution. This capability could, in turn, lead to improvements in ArM efficiency similar to those obtained for natural enzymes, providing systems suitable for practical applications and greater insight into the role of second coordination sphere interactions in organometallic catalysis. Since its renaissance in the early 2000’s, different aspects of artificial metalloenzymes have been extensively reviewed and highlighted. Our intent is to provide a comprehensive overview of all work in the field up to December 2016, organized according to reaction class. Because of the wide range of non-natural reactions catalyzed by ArMs, this was done using a functional-group transformation classification. The review begins with a summary of the proteins and the anchoring strategies used to date for the creation of ArMs, followed by a historical perspective. Then follows a summary of the reactions catalyzed by ArMs and a concluding critical outlook. This analysis allows for comparison of similar reactions catalyzed by ArMs constructed using different metallocofactor anchoring strategies, cofactors, protein scaffolds, and mutagenesis strategies. These data will be used to construct a searchable Web site on ArMs that will be updated regularly by the authors.


Notes: ---

Beyond the Second Coordination Sphere: Engineering Dirhodium Artificial Metalloenzymes To Enable Protein Control of Transition Metal Catalysis

Review

Lewis, J.C.

Acc. Chem. Res. 2019, 52, 576-584, 10.1021/acs.accounts.8b00625

Transition metal catalysis is a powerful tool for chemical synthesis, a standard by which understanding of elementary chemical processes can be measured, and a source of awe for those who simply appreciate the difficulty of cleaving and forming chemical bonds. Each of these statements is amplified in cases where the transition metal catalyst controls the selectivity of a chemical reaction. Enantioselective catalysis is a challenging but well-established phenomenon, and regio- or site-selective catalysis is increasingly common. On the other hand, transition-metal-catalyzed reactions are typically conducted under highly optimized conditions. Rigorous exclusion of air and water is common, and it is taken for granted that only a single substrate (of a particular class) will be present in a reaction, a desired site selectivity can be achieved by installing a directing group, and undesired reactivity can be blocked with protecting groups. These are all reasonable synthetic strategies, but they also highlight limits to catalyst control. The utility of transition metal catalysis could be greatly expanded if catalysts possessed the ability to regulate which molecules they encounter and the relative orientation of those molecules. The rapid and widespread adoption of stoichiometric bioorthogonal reactions illustrates the utility of robust reactions that proceed with high selectivity and specificity under mild reaction conditions. Expanding this capability beyond preprogrammed substrate pairs via catalyst control could therefore have an enormous impact on molecular science. Many metalloenzymes exhibit this level of catalyst control, and directed evolution can be used to rapidly improve the catalytic properties of these systems. On the other hand, the range of reactions catalyzed by enzymes is limited relative to that developed by chemists. The possibility of imparting enzyme-like activity, selectivity, and evolvability to reactions catalyzed by synthetic transition metal complexes has inspired the creation of artificial metalloenzymes (ArMs). The increasing levels of catalyst control exhibited by ArMs developed to date suggest that these systems could constitute a powerful platform for bioorthogonal transition metal catalysis and for selective catalysis in general. This Account outlines the development of a new class of ArMs based on a prolyl oligopeptidase (POP) scaffold. Studies conducted on POP ArMs containing a covalently linked dirhodium cofactor have shown that POP can impart enantioselectivity to a range of dirhodium-catalyzed reactions, increase reaction rates, and improve the specificity for reaction of dirhodium carbene intermediates with targeted organic substrates over components of cell lysate, including bulk water. Several design features of these ArMs enabled their evolution via random mutagenesis, which revealed that mutations throughout the POP scaffold, beyond the second sphere of the dirhodium cofactor, were important for ArM activity and selectivity. While it was anticipated that the POP scaffold would be capable of encapsulating and thus controlling the selectivity of bulky cofactors, molecular dynamics studies also suggest that POP conformational dynamics plays a role in its unique efficacy. These advances in scaffold selection, bioconjugation, and evolution form the basis of our ongoing efforts to control transition metal reactivity using protein scaffolds with the goal of enabling unique synthetic capabilities, including bioorthogonal catalysis.


Notes: ---

Beyond the Second Coordination Sphere: Engineering Dirhodium Artificial Metalloenzymes To Enable Protein Control of Transition Metal Catalysis

Review

Lewis, J.C.

Acc. Chem. Res. 2019, 52, 576-584, 10.1021/acs.accounts.8b00625

Transition metal catalysis is a powerful tool for chemical synthesis, a standard by which understanding of elementary chemical processes can be measured, and a source of awe for those who simply appreciate the difficulty of cleaving and forming chemical bonds. Each of these statements is amplified in cases where the transition metal catalyst controls the selectivity of a chemical reaction. Enantioselective catalysis is a challenging but well-established phenomenon, and regio- or siteselective catalysis is increasingly common. On the other hand, transition-metal-catalyzed reactions are typically conducted under highly optimized conditions. Rigorous exclusion of air and water is common, and it is taken for granted that only a single substrate (of a particular class) will be present in a reaction, a desired site selectivity can be achieved by installing a directing group, and undesired reactivity can be blocked with protecting groups. These are all reasonable synthetic strategies, but they also highlight limits to catalyst control. The utility of transition metal catalysis could be greatly expanded if catalysts possessed the ability to regulate which molecules they encounter and the relative orientation of those molecules. The rapid and widespread adoption of stoichiometric bioorthogonal reactions illustrates the utility of robust reactions that proceed with high selectivity and specificity under mild reaction conditions. Expanding this capability beyond preprogrammed substrate pairs via catalyst control could therefore have an enormous impact on molecular science. Many metalloenzymes exhibit this level of catalyst control, and directed evolution can be used to rapidly improve the catalytic properties of these systems. On the other hand, the range of reactions catalyzed by enzymes is limited relative to that developed by chemists. The possibility of imparting enzyme-like activity, selectivity, and evolvability to reactions catalyzed by synthetic transition metal complexes has inspired the creation of artificial metalloenzymes (ArMs). The increasing levels of catalyst control exhibited by ArMs developed to date suggest that these systems could constitute a powerful platform for bioorthogonal transition metal catalysis and for selective catalysis in general. This Account outlines the development of a new class of ArMs based on a prolyl oligopeptidase (POP) scaffold. Studies conducted on POP ArMs containing a covalently linked dirhodium cofactor have shown that POP can impart enantioselectivity to a range of dirhodium-catalyzed reactions, increase reaction rates, and improve the specificity for reaction of dirhodium carbene intermediates with targeted organic substrates over components of cell lysate, including bulk water. Several design features of these ArMs enabled their evolution via random mutagenesis, which revealed that mutations throughout the POP scaffold, beyond the second sphere of the dirhodium cofactor, were important for ArM activity and selectivity. While it was anticipated that the POP scaffold would be capable of encapsulating and thus controlling the selectivity of bulky cofactors, molecular dynamics studies also suggest that POP conformational dynamics plays a role in its unique efficacy. These advances in scaffold selection, bioconjugation, and evolution form the basis of our ongoing efforts to control transition metal reactivity using protein scaffolds with the goal of enabling unique synthetic capabilities, including bioorthogonal catalysis.


Notes: ---

Engineering a Dirhodium Artificial Metalloenzyme for Selective Olefin Cyclopropanation

Lewis, J.C.

Nat. Commun. 2015, 6, 10.1038/ncomms8789

Artificial metalloenzymes (ArMs) formed by incorporating synthetic metal catalysts into protein scaffolds have the potential to impart to chemical reactions selectivity that would be difficult to achieve using metal catalysts alone. In this work, we covalently link an alkyne-substituted dirhodium catalyst to a prolyl oligopeptidase containing a genetically encoded L-4-azidophenylalanine residue to create an ArM that catalyses olefin cyclopropanation. Scaffold mutagenesis is then used to improve the enantioselectivity of this reaction, and cyclopropanation of a range of styrenes and donor–acceptor carbene precursors were accepted. The ArM reduces the formation of byproducts, including those resulting from the reaction of dirhodium–carbene intermediates with water. This shows that an ArM can improve the substrate specificity of a catalyst and, for the first time, the water tolerance of a metal-catalysed reaction. Given the diversity of reactions catalysed by dirhodium complexes, we anticipate that dirhodium ArMs will provide many unique opportunities for selective catalysis.


Metal: Rh
Ligand type: Poly-carboxylic acid
Anchoring strategy: Covalent
Optimization: Chemical & genetic
Reaction: Cyclopropanation
Max TON: 74
ee: 92
PDB: ---
Notes: ---

Evolving Artificial Metalloenzymes via Random Mutagenesis

Lewis, J.C.

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.


Metal: Rh
Ligand type: OAc
Anchoring strategy: Covalent
Optimization: Chemical & genetic
Reaction: Cyclopropanation
Max TON: 66
ee: 94
PDB: 5T88
Notes: Mutagenesis of the ArM by error-prone PCR

Metal: Rh
Ligand type: OAc
Anchoring strategy: Covalent
Optimization: Chemical & genetic
Reaction: N-H Insertion
Max TON: 73
ee: 40
PDB: 5T88
Notes: Mutagenesis of the ArM by error-prone PCR

Metal: Rh
Ligand type: OAc
Anchoring strategy: Covalent
Optimization: Chemical & genetic
Reaction: S-H insertion
Max TON: 64
ee: 32
PDB: 5T88
Notes: Mutagenesis of the ArM by error-prone PCR

Metal: Rh
Ligand type: OAc
Anchoring strategy: Covalent
Optimization: Chemical & genetic
Reaction: Si-H insertion
Max TON: 35
ee: 64
PDB: 5T88
Notes: Mutagenesis of the ArM by error-prone PCR

Manganese Terpyridine Artificial Metalloenzymes for Benzylic Oxygenation and Olefin Epoxidation

Lewis, J.C.

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: Mn
Ligand type: Poly-pyridine
Host protein: Nitrobindin (Nb)
Anchoring strategy: Covalent
Optimization: Chemical
Max TON: 19.2
ee: ---
PDB: 3EMM
Notes: ---

Metal: Mn
Ligand type: Poly-pyridine
Host protein: Nitrobindin (Nb)
Anchoring strategy: Covalent
Optimization: Chemical
Reaction: Epoxidation
Max TON: 19.8
ee: ---
PDB: 3EMM
Notes: ---

Metallopeptide Catalysts and Artificial Metalloenzymes Containing Unnatural Amino Acids

Review

Lewis, J.C.

Curr. Opin. Chem. Biol. 2015, 25, 27-35, 10.1016/j.cbpa.2014.12.016

Metallopeptide catalysts and artificial metalloenzymes built from peptide scaffolds and catalytically active metal centers possess a number of exciting properties that could be exploited for selective catalysis. Control over metal catalyst secondary coordination spheres, compatibility with library based methods for optimization and evolution, and biocompatibility stand out in this regard. A wide range of unnatural amino acids (UAAs) have been incorporated into peptide and protein scaffolds using several distinct methods, and the resulting UAAs containing scaffolds can be used to create novel hybrid metal–peptide catalysts. Promising levels of selectivity have been demonstrated for several hybrid catalysts, and these provide a strong impetus and important lessons for the design of and optimization of hybrid catalysts.


Notes: ---

Preparation of Artificial Metalloenzymes

Review

Lewis, J.C.

Artificial Metalloenzymes and MetalloDNAzymes in Catalysis: From Design to Applications 2018, 1-40, 10.1002/9783527804085.ch1

Transition metal catalysts and enzymes are ubiquitous tools for chemical synthesis. Potential benefits of combining complementary properties of these catalysts have driven efforts to create artificial metalloenzymes (ArMs), hybrid constructs comprised of synthetic metal centers embedded within protein scaffolds. This unique composition necessitates the use of synthetic chemistry, bioconjugation methodology, and protein engineering for ArM formation. Despite this challenge, a range of approaches for ArM formation has been developed. This chapter provides an overview of these different approaches and discussion of potential advantages and disadvantages of each.


Notes: Book chapter

Selective C–H Bond Functionalization Using Repurposed or Artificial Metalloenzymes

Review

Lewis, J.C.

Curr. Opin. Chem. Biol. 2017, 37, 48-55, 10.1016/j.cbpa.2016.12.027

Catalytic CH bond functionalization has become an important tool for organic synthesis. Metalloenzymes offer a solution to one of the foremost challenges in this field, site-selective CH functionalization, but they are only capable of catalyzing a subset of the CH functionalization reactions known to small molecule catalysts. To overcome this limitation, metalloenzymes have been repurposed by exploiting the reactivity of their native cofactors toward substrates not found in nature. Additionally, new reactivity has been accessed by incorporating synthetic metal cofactors into protein scaffolds to form artificial metalloenzymes. The selectivity and activity of these catalysts has been tuned using directed evolution. This review covers the recent progress in developing and optimizing both repurposed and artificial metalloenzymes as catalysts for selective CH bond functionalization.


Notes: ---