Despite the astonishing breadth of enzymes in nature, no enzymes are known for many of the valuable catalytic transformations discovered by chemists. Recent work in enzyme design and evolution, ...however, gives us good reason to think that this will change. We describe a chemomimetic biocatalysis approach that draws from small-molecule catalysis and synthetic chemistry, enzymology, and molecular evolution to discover or create enzymes with non-natural reactivities. We illustrate how cofactor-dependent enzymes can be exploited to promote reactions first established with related chemical catalysts. The cofactors can be biological, or they can be non-biological to further expand catalytic possibilities. The ability of enzymes to amplify and precisely control the reactivity of their cofactors together with the ability to optimize non-natural reactivity by directed evolution promises to yield exceptional catalysts for challenging transformations that have no biological counterparts.
Chiral 1,2‐amino alcohols are widely represented in biologically active compounds from neurotransmitters to antivirals. While many synthetic methods have been developed for accessing amino alcohols, ...the direct aminohydroxylation of alkenes to unprotected, enantioenriched amino alcohols remains a challenge. Using directed evolution, we have engineered a hemoprotein biocatalyst based on a thermostable cytochrome c that directly transforms alkenes to amino alcohols with high enantioselectivity (up to 2500 TTN and 90 % ee) under anaerobic conditions with O‐pivaloylhydroxylamine as an aminating reagent. The reaction is proposed to proceed via a reactive iron‐nitrogen species generated in the enzyme active site, enabling tuning of the catalyst's activity and selectivity by protein engineering.
Go direct: A hemoprotein catalyst was engineered to transform alkenes directly to amino alcohols with high enantioselectivity. Derived by directed evolution from a thermostable cytochrome c, the protein catalyst uses O‐pivaloylhydroxylamine to generate a reactive iron‐nitrogen species.
Redox enzymes offer many powerful transformations for the efficient industrial-scale synthesis of diverse chemicals desired by society. Here we survey recent preparative applications of redox ...enzymes, highlighting both mature enzyme platforms and promising technologies for future applications. While in some cases commercial enzymes can be employed directly on industrial scales, in other cases protein engineering is necessary to evolve an enzyme fit for non-biological substrates and conditions. Both approaches require the input of process engineering to properly balance the needs of the enzymatic chemistry with the requirements for an industrial process. A convergence of advances in enzyme discovery, protein engineering, and process engineering is expected to fuel a more rapid development of enzymatic synthetic processes and a wider adoption of biocatalysis on industrial scales.
C-H bonds are ubiquitous structural units of organic molecules. Although these bonds are generally considered to be chemically inert, the recent emergence of methods for C-H functionalization ...promises to transform the way synthetic chemistry is performed. The intermolecular amination of C-H bonds represents a particularly desirable and challenging transformation for which no efficient, highly selective, and renewable catalysts exist. Here we report the directed evolution of an iron-containing enzymatic catalyst-based on a cytochrome P450 monooxygenase-for the highly enantioselective intermolecular amination of benzylic C-H bonds. The biocatalyst is capable of up to 1,300 turnovers, exhibits excellent enantioselectivities, and provides access to valuable benzylic amines. Iron complexes are generally poor catalysts for C-H amination: in this catalyst, the enzyme's protein framework confers activity on an otherwise unreactive iron-haem cofactor.
Serendipity has long been a welcome yet elusive phenomenon in the advancement of chemistry. We sought to exploit serendipity as a means of rapidly identifying unanticipated chemical transformations. ...By using a high-throughput, automated workflow and evaluating a large number of random reactions, we have discovered a photoredox-catalyzed C-H arylation reaction for the construction of benzylic amines, an important structural motif within pharmaceutical compounds that is not readily accessed via simple substrates. The mechanism directly couples tertiary amines with cyanoaromatics by using mild and operationally trivial conditions.
Sigmatropic rearrangements, while rare in biology, offer opportunities for the efficient and selective synthesis of complex chemical motifs. A “P411” serine‐ligated variant of cytochrome P450BM3 has ...been engineered to initiate a sulfimidation/2,3‐sigmatropic rearrangement sequence in whole E. coli cells, a non‐natural function for any enzyme, providing access to enantioenriched, protected allylic amines. Five mutations in the enzyme substantially enhance its activity toward this new function, demonstrating the evolvability of the catalyst toward challenging nitrene transfer reactions. The evolved catalyst additionally performs the highly enantioselective imidation of non‐allylic sulfides.
A serine‐ligated variant of cytochrome P450BM3 has been engineered to initiate a sulfimidation/2,3‐sigmatropic rearrangement sequence in whole E. coli cells, providing access to enantioenriched, protected allylic amines. The results highlight the ability of enzymes to adapt, through directed evolution, to facilitate valuable reaction pathways for which no natural enzymes have evolved.
The direct α-heteroarylation of tertiary amines has been accomplished
photoredox catalysis to generate valuable benzylic amine pharmacophores. A variety of five-and six-membered chloroheteroarenes ...are shown to function as viable coupling partners for the α-arylation of a diverse range of cyclic and acyclic amines. Evidence is provided for a homolytic aromatic substitution mechanism, in which a catalyticallygenerated α-amino radical undergoes direct addition to an electrophilic chloroarene.
The repurposing of hemoproteins for non-natural carbene transfer activities has generated enzymes for functions previously accessible only to chemical catalysts. With activities constrained to ...specific substrate classes, however, the synthetic utility of these new biocatalysts has been limited. To expand the capabilities of non-natural carbene transfer biocatalysis, we engineered variants of Cytochrome P450BM3 that catalyze the cyclopropanation of heteroatom-bearing alkenes, providing valuable nitrogen-, oxygen-, and sulfur-substituted cyclopropanes. Four or five active-site mutations converted a single parent enzyme into selective catalysts for the synthesis of both cis and trans heteroatom-substituted cyclopropanes, with high diastereoselectivities and enantioselectivities and up to 40 000 total turnovers. This work highlights the ease of tuning hemoproteins by directed evolution for efficient cyclopropanation of new substrate classes and expands the catalytic functions of iron heme proteins.
The formation of C-N bonds-of great importance to the pharmaceutical industry-can be facilitated enzymatically using nucleophilic and nitrene transfer mechanisms. However, neither natural nor ...engineered enzymes are known to generate and control nitrogen-centred radicals, which serve as valuable species for C-N bond formation. Here we use flavin-dependent 'ene'-reductases with an exogenous photoredox catalyst to selectively generate amidyl radicals within the protein active site. These enzymes are engineered through directed evolution to catalyse 5-exo, 6-endo, 7-endo, 8-endo, and intermolecular hydroamination reactions with high levels of enantioselectivity. Mechanistic studies suggest that radical initiation occurs via an enzyme-gated mechanism, where the protein thermodynamically activates the substrate for reduction by the photocatalyst. Molecular dynamics studies indicate that the enzymes bind substrates using non-canonical binding interactions, which may serve as a handle to further manipulate reactivity. This approach demonstrates the versatility of these enzymes for controlling the reactivity of high-energy radical intermediates and highlights the opportunity for synergistic catalyst strategies to unlock previously inaccessible enzymatic functions.
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