Directed Evolution of a Fe(II)- and α-Ketoglutarate-Dependent Dioxygenase for Site-Selective Azidation of Unactivated Aliphatic C-H Bonds

Gomez, Christian A.; Mondal, Dibyendu; Du, Qian; Chan, Natalie H.; Lewis, Jared C.

doi:10.26434/chemrxiv-2022-ddfzp

Cited by 3 publications

(3 citation statements)

References 47 publications

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“…Without such bias, the only demonstration of a selective, catalyst-controlled derivatization is the seminal report by Davies and co-workers − on rhodium-catalyzed asymmetric carbene insertion into hydrocarbon C–H bonds (also see ref ). Recently, selective azidation and nitration of unactivated C–H bonds have been demonstrated with non-heme iron enzymes. − Here, we establish that P450 enzymes can be engineered for the introduction of nitrogen at unbiased, unactivated C(sp 3 )–H centers with high site and stereoselectivity, revealing a new biochemical pathway for C–H functionalization that, analogous to P450-catalyzed C–H hydroxylation, can be tuned and diversified by evolution (Figure ).…”

Section: Introductionmentioning

confidence: 59%

Enzymatic Nitrogen Insertion into Unactivated C–H Bonds

et al. 2022

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Selective functionalization of aliphatic C–H bonds, ubiquitous in molecular structures, could allow ready access to diverse chemical products. While enzymatic oxygenation of C–H bonds is well established, the analogous enzymatic nitrogen functionalization is still unknown; nature is reliant on preoxidized compounds for nitrogen incorporation. Likewise, synthetic methods for selective nitrogen derivatization of unbiased C–H bonds remain elusive. In this work, new-to-nature heme-containing nitrene transferases were used as starting points for the directed evolution of enzymes to selectively aminate and amidate unactivated C(sp3)–H sites. The desymmetrization of methyl- and ethylcyclohexane with divergent site selectivity is offered as demonstration. The evolved enzymes in these lineages are highly promiscuous and show activity toward a wide array of substrates, providing a foundation for further evolution of nitrene transferase function. Computational studies and kinetic isotope effects (KIEs) are consistent with a stepwise radical pathway involving an irreversible, enantiodetermining hydrogen atom transfer (HAT), followed by a lower-barrier diastereoselectivity-determining radical rebound step. In-enzyme molecular dynamics (MD) simulations reveal a predominantly hydrophobic pocket with favorable dispersion interactions with the substrate. By offering a direct path from saturated precursors, these enzymes present a new biochemical logic for accessing nitrogen-containing compounds.

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Section: Introductionmentioning

confidence: 59%

Enzymatic Nitrogen Insertion into Unactivated C–H Bonds

et al. 2022

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“…Recently, selective azidation and nitration of unactivated C-H bonds has been demonstrated with non-heme iron enzymes. [33][34][35] Here, we establish that P450 enzymes can be engineered for introduction of nitrogen at unbiased, unactivated C(sp 3 )-H centers with high site and stereoselectivity, revealing a new biochemical pathway for C-H functionalization that, analogous to P450-catalyzed C-H hydroxylation, can be tuned and diversified by evolution (Figure 1).…”

Section: Introductionmentioning

confidence: 75%

Enzymatic Nitrogen Insertion into Unactivated C–H Bonds

Athavale

Gao

Das

et al. 2022

Preprint

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Selective functionalization of aliphatic C–H bonds, ubiquitous in molecular structures, could allow ready access to diverse chemical products. While enzymatic oxygenation of C–H bonds is well established, the analogous enzymatic nitrogen functionalization is still unknown; nature is reliant on pre-oxidized compounds for nitrogen incorporation. Likewise, synthetic methods for selective nitrogen derivatization of unbiased C–H bonds remain elusive. In this work, new-to-nature heme-containing nitrene transferases were used as starting points for the directed evolution of enzymes to selectively aminate and amidate unactivated C(sp3)–H sites. The desymmetrization of methyl- and ethylcyclohexane with divergent site selectivity is offered as demonstration. The evolved enzymes in these lineages are highly promiscuous and show activity towards a wide array of substrates, providing a foundation for further evolution of nitrene transferase function. Computational studies and kinetic isotope effects (KIEs) are consistent with a stepwise radical pathway involving an irreversible, enantiodetermining hydrogen atom transfer (HAT), followed by a lower-barrier diastereoselectivity determining radical rebound step. In-enzyme molecular dynamics (MD) simulations reveal a predominantly hydrophobic pocket with favorable dispersion interactions with the substrate. By offering a direct path from saturated precursors, these enzymes present a new biochemical logic for accessing nitrogen-containing compounds.

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“…6 Members of the latter three classes exhibit catalyst-controlled site-selectivity to enable precise placement of halogen substituents in a diverse range of substrates. The vanadium and non-heme iron systems also catalyze stereoselective halogenation via olefin halocyclization 7,8 and aliphatic C-H functionalization 9,10 , respectively.…”

Section: Introductionmentioning

confidence: 99%

Asymmetric Catalysis by Flavin Dependent Halogenases

Jiang

Lewis

2022

Preprint

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In nature, flavin dependent halogenases (FDHs) catalyze site-selective chlorination and bromination of aromatic natural products. This ability has led to extensive efforts to engineer FDHs for selective chlorination, bromination, and iodination of electron rich aromatic compounds. On the other hand, FDHs are unique among halogenases and haloperoxidases that exhibit catalyst-controlled site selectivity in that no examples of enantioselective FDH catalysis in natural product biosynthesis have been characterized. Over the past several years, our group has established that FDHs can catalyze enantioselective reactions involving desymmetrization, atroposelective halogenation, and halocyclization. Achieving high activity and selectivity for these reactions has required extensive mutagenesis and mitigation of problems resulting from hypohalous acid generated during FDH catalysis. The single-component flavin reductase/FDH AetF is unique among the wild type enzyme, we have studied in that it provides high activity and selectivity toward several asymmetric transformations. These results highlight the ability of FDH active sites to tolerate different substrate topologies and suggest that they could be useful for a broad range of oxidative halogenations.

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Directed Evolution of a Fe(II)- and α-Ketoglutarate-Dependent Dioxygenase for Site-Selective Azidation of Unactivated Aliphatic C-H Bonds

Cited by 3 publications

References 47 publications

Enzymatic Nitrogen Insertion into Unactivated C–H Bonds

Enzymatic Nitrogen Insertion into Unactivated C–H Bonds

Enzymatic Nitrogen Insertion into Unactivated C–H Bonds

Asymmetric Catalysis by Flavin Dependent Halogenases

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