Initiating and terminating free-radical reactionis via hydrogen atom transfer (HAT) is an attractive means of avoiding substrate prefunctionalization. Small molecule catalysts and reagents, however, ...struggle to execute this fundamental step with useful levels of diastereoselectivity and enantioselectivity. In contrast, nature often carries out HAT with exquisite levels of selectivity for even electronically unactivated carbon–hydrogen bonds. By understanding how enzymes exploit and control this fundamental step, new strategies can be developed to address several long-standing challenges in free-radical reactions. This review will cover recent discoveries in biocatalysis that exploit a HAT mechanism to either initiate or terminate novel one-electron reactions.
Photoenzymatic intermolecular hydroalkylations of olefins are highly enantioselective for chiral centers formed during radical termination but poorly selective for centers set in the C–C bond-forming ...event. Here, we report the evolution of a flavin-dependent “ene”-reductase to catalyze the coupling of α,α-dichloroamides with alkenes to afford α-chloroamides in good yield with excellent chemo- and stereoselectivity. These products can serve as linchpins in the synthesis of pharmaceutically valuable motifs. Mechanistic studies indicate that radical formation occurs by exciting a charge-transfer complex templated by the protein. Precise control over the orientation of molecules within the charge-transfer complex potentially accounts for the observed stereoselectivity. The work expands the types of motifs that can be prepared using photoenzymatic catalysis.
Strategies that provide enzymes with the ability to catalyse non-natural reactions are of considerable synthetic value. Photoredox catalysis has proved adept at expanding the synthetic repertoire of ...existing catalytic platforms, yet, in the realm of biocatalysis it has primarily been used for cofactor regeneration. Here we show that photoredox catalysts can be used to enable new catalytic function in nicotinamide-dependent enzymes. Under visible-light irradiation, xanthene-based photocatalysts enable a double-bond reductase to catalyse an enantioselective deacetoxylation. Mechanistic experiments support the intermediacy of an α-acyl radical, formed after the elimination of acetate. Isotopic labelling experiments support nicotinamide as the source of the hydrogen atom. Preliminary calculations and mechanistic experiments suggest that binding to the protein attenuates the reduction potential of the starting material, an important feature for localizing radical formation to the enzyme active site. The generality of this approach is highlighted with the radical dehalogenation of α-bromoamides catalysed by ketoreductases with Eosin Y as a photocatalyst.
Flavin‐dependent ‘ene’‐reductases (EREDs) are highly selective catalysts for the asymmetric reduction of activated alkenes. This function is, however, limited to enones, enoates, and nitroalkenes ...using the native hydride transfer mechanism. Here we demonstrate that EREDs can reduce vinyl pyridines when irradiated with visible light in the presence of a photoredox catalyst. Experimental evidence suggests the reaction proceeds via a radical mechanism where the vinyl pyridine is reduced to the corresponding neutral benzylic radical in solution. DFT calculations reveal this radical to be “dynamically stable”, suggesting it is sufficiently long‐lived to diffuse into the enzyme active site for stereoselective hydrogen atom transfer. This reduction mechanism is distinct from the native one, highlighting the opportunity to expand the synthetic capabilities of existing enzyme platforms by exploiting new mechanistic models.
HAT‐trick: Flavin‐dependent ‘ene'‐reductases can reduce vinyl pyridines when irradiated with visible light in the presence of a photoredox catalyst. The reaction proceeds via a radical mechanism where the vinyl pyridine is reduced to a neutral benzylic radical in solution. Calculations reveal this radical to be “dynamically stable” and therefore sufficiently long‐lived to diffuse into the enzyme active site for stereoselective hydrogen atom transfer (HAT).
Flavin‐dependent ene‐reductases (EREDs) are known to stereoselectively reduce activated alkenes, but are inactive toward carbonyls. Demonstrated here is that in the presence of photoredox catalysts, ...these enzymes will reduce aromatic ketones. Mechanistic experiments suggest this reaction proceeds through ketyl radical formation, a reaction pathway that is distinct from the native hydride‐transfer mechanism. Furthermore, this reactivity is accessible without modification of either the enzyme or cofactors, allowing both native and non‐natural mechanisms to occur simultaneously. Based on control experiments, we hypothesize that binding to the enzyme active site attenuates the reduction potential of the substrate, enabling single‐electron reduction. This reactivity highlights opportunities to access new catalytic manifolds by merging photoredox catalysis with biocatalysis.
Flavin misbehavin′: Merging photocatalysis with biocatalysis leads to new chemical reactivity for flavin‐dependent ene‐reductases. Mechanistic studies support a mechanism in which enantioenriched alcohols are produced by hydrogen‐atom transfer from flavin to the ketyl radical.
Engineering enzymes with novel reaction modes promises to expand the applications of biocatalysis in chemical synthesis and will enhance our understanding of how enzymes acquire new functions. The ...insertion of nitrogen-containing functional groups into unactivated C–H bonds is not catalyzed by known enzymes but was recently demonstrated using engineered variants of cytochrome P450BM3 (CYP102A1) from Bacillus megaterium. Here, we extend this novel P450-catalyzed reaction to include intermolecular insertion of nitrogen into thioethers to form sulfimides. An examination of the reactivity of different P450BM3 variants toward a range of substrates demonstrates that electronic properties of the substrates are important in this novel enzyme-catalyzed reaction. Moreover, amino acid substitutions have a large effect on the rate and stereoselectivity of sulfimidation, demonstrating that the protein plays a key role in determining reactivity and selectivity. These results provide a stepping stone for engineering more complex nitrogen-atom-transfer reactions in P450 enzymes and developing a more comprehensive biocatalytic repertoire.
Non-natural photoenzymatic reactions reported to date have depended on the excitation of electron donor–acceptor complexes formed between substrates and cofactors within protein active sites to ...facilitate electron transfer. While this mechanism has unlocked new reactivity, it limits the types of substrates that can be involved in this area of catalysis. Here we demonstrate that direct excitation of flavin hydroquinone within “ene”-reductase active sites enables new substrates to participate in photoenzymatic reactions. We found that by using photoexcitation these enzymes gain the ability to reduce acrylamides through a single electron transfer mechanism.
Photoenzymes are biological catalysts that use light to convert starting materials into products. These catalysts require photon absorption for each turnover, making quantum efficiency an important ...optimization parameter. Flavin‐dependent “ene”‐reductases (EREDs) display latent photoenzymatic activity for synthetically valuable hydroalkylations; however, protein engineering has not been used to optimize this non‐natural function. We describe a protein engineering platform for the high throughput optimization of photoenzymes. A single round of engineering results in improved catalytic function toward the synthesis of γ, δ, ϵ‐lactams, and acyclic amides. Mechanistic studies show that key mutations can alter the enzyme's excited state dynamics, enhance its photon efficiency, and ultimately increase catalyst performance. Transient absorption spectroscopy reveals that engineered variants display dramatically decreased radical lifetimes, indicating an evolution toward a concerted mechanism.
We developed a novel HTS engineering platform to optimize photoenzymatic activity. The improvements in variants were correlated to an increase in enzymatic photon efficiency. Transient absorption spectroscopy revealed a shift from a stepwise to a concerted mechanism. The platform was expanded to improve the synthesis of γ, δ, ϵ‐lactams, and acyclic amides.
It's all under control: The selection between CC and CH activation pathways in 8‐acylquinolines can be influenced by the choice of catalyst and solvent (see scheme; cod=cycloocta‐1,5‐diene). In ...this approach, an unstrained CC σ bond is activated and subsequent intermolecular carboacylation of an olefin results in the formation of two new CC σ bonds.
PDF
