Selective biocatalytic hydroxylation of unactivated methylene C–H bonds in cyclic alkyl substrates

Sarkar, Md. Raihan; Dasgupta, Samrat; Pyke, Simon M.; Bell, Stephen

doi:10.1039/c9cc02060h

Cited by 14 publications

(15 citation statements)

References 34 publications

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“…Under these conditions, this variant was found to produce the desired sultam product 1b in 70% yield with a TTN value of 14 800 (Table , Entry 17). This TTN value corresponds to the highest catalytic activity for a biocatalytic nitrene transfer reaction reported to date, with only one notable exception, and it compares well with the highest TTN values reported for monooxygenation reactions catalyzed by engineered P450s. − …”

Section: Results and Discussionsupporting

confidence: 75%

“…Arnold and co-workers reported a comparable activity (∼360 TON) for serine-ligated P450 BM3 variants in the same reaction, and a similar performance was observed for artificial P450 metalloenzymes harboring an abiological Ir(Me) mesoporphyrin IX cofactor (∼300 TON) . While the catalytic activity of these engineered P450s in C–H amination reactions exceeds that of synthetic catalysts (typically, <100–150 TON), this reactivity remains far below that achievable by these enzymes in monooxygenation reactions, some of which have been reported to exceed 60 000 total turnovers. − …”

Section: Introductionmentioning

confidence: 77%

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Mechanism-Guided Design and Discovery of Efficient Cytochrome P450-Derived C–H Amination Biocatalysts

et al. 2020

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Cytochromes P450 have been recently identified as a promising class of biocatalysts for mediating C–H aminations via nitrene transfer, a valuable transformation for forging new C–N bonds. The catalytic efficiency of P450s in these non-native transformations is however significantly inferior to that exhibited by these enzymes in their native monooxygenase function. Using a mechanism-guided strategy, we report here the rational design of a series of P450BM3-based variants with dramatically enhanced C–H amination activity acquired through disruption of the native proton relay network and other highly conserved structural elements within this class of enzymes. This approach further guided the identification of XplA and BezE, two “atypical” natural P450s implicated in the degradation of a man-made explosive and in benzastatins biosynthesis, respectively, as very efficient C–H aminases. Both XplA and BezE could be engineered to further improve their C–H amination reactivity, which demonstrates their evolvability for abiological reactions. These engineered and natural P450 catalysts can promote the intramolecular C–H amination of arylsulfonyl azides with over 10 000–14 000 catalytic turnovers, ranking among the most efficient nitrene transfer biocatalysts reported to date. Mechanistic and structure–reactivity studies provide insights into the origin of the C–H amination reactivity enhancement and highlight the divergent structural requirements inherent to supporting C–H amination versus C–H monooxygenation reactivity within this class of enzymes. Overall, this work provides new promising scaffolds for the development of nitrene transferases and demonstrates the value of mechanism-driven rational design as a strategy for improving the catalytic efficiency of metalloenzymes in the context of abiological transformations.

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Section: Results and Discussionsupporting

confidence: 75%

Section: Introductionmentioning

confidence: 77%

Mechanism-Guided Design and Discovery of Efficient Cytochrome P450-Derived C–H Amination Biocatalysts

et al. 2020

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“…[53] More recently, Bell et al, used CYP101B1 to oxidize cyclic hydrocarbons with the ester anchoring group modified the ring structures. [54] Interestingly, different ester anchoring groups (e. g. acetate, butyrate, isobutyrate) imparted different enhanced effect on the enzymatic conversion, while all hydroxylation occurred specifically at CÀ H bounds on the opposite side of the ring to the ester or ketone moiety. These results highlighted the usefulness of the d/p group to improve the regioselectivity and efficiency of P450 catalyzed transformations.…”

Section: Substrate Engineeringmentioning

confidence: 99%

Harnessing P450 Enzyme for Biotechnology and Synthetic Biology

Zhang

Wang

2021

ChemBioChem

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Cytochrome P450 enzymes (P450s, CYPs) catalyze the oxidative transformation of a wide range of organic substrates. Their functions are crucial to xenobiotic metabolism and steroid transformation in humans and other organisms. The enzymes are promising for synthetic biology applications but limited by several drawbacks including low turnover rates, poor stability, the dependance of expensive cofactors and redox partners, and the narrow substrate scope. To conquer these obstacles, emerging strategies including substrate engineering, usage of decoy and decoy‐based small molecules auxiliaries, designing of artificial enzyme cascades and the incorporation of materials have been explored based on the unique properties of P450s. These strategies can be applied to a wide range of P450s and can be combined with protein engineering to improve the enzymatic activities. This minireview will focus on some recent developments of these strategies which have been used to leverage P450 catalysis. Remaining challenges and future opportunities will also be discussed.

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“…The group investigated using ester protecting groups as chemical auxiliaries and directing groups for the biocatalytic hydroxylation, hypothesising these may mimic chemical features of CYP101B1’s known substrates, the norisoprenoids. Hydroxylation of cyclooctyl acetate by CYP101B1 generated trans‐ 5‐hydroxycyclooctyl in >95 % yield (Figure 1B) [39] . Additionally, selective sp 3 C−H hydroxylation may be useful for the functionalisation of fatty acids for pharmaceutical applications.…”

Section: Oxygenating Biocatalystsmentioning

confidence: 99%

“…[38] rings. [39] The group investigated using ester protecting groups as chemical auxiliaries and directing groups for the biocatalytic hydroxylation, hypothesising these may mimic chemical features of CYP101B1's known substrates, the norisoprenoids. Hydroxylation of cyclooctyl acetate by CYP101B1 generated trans-5-hydroxycyclooctyl in > 95 % yield (Figure 1B).…”

Section: Cytochrome P450smentioning

confidence: 99%

Oxygenating Biocatalysts for Hydroxyl Functionalisation in Drug Discovery and Development

Charlton

Hayes

2022

ChemMedChem

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CÀ H oxyfunctionalisation remains a distinct challenge for synthetic organic chemists. Oxygenases and peroxygenases (grouped here as "oxygenating biocatalysts") catalyse the oxidation of a substrate with molecular oxygen or hydrogen peroxide as oxidant. The application of oxygenating biocatalysts in organic synthesis has dramatically increased over the last decade, producing complex compounds with potential uses in the pharmaceutical industry. This review will focus on hydroxyl functionalisation using oxygenating biocatalysts as a tool for drug discovery and development. Established oxygenating biocatalysts, such as cytochrome P450s and flavin-dependent monooxygenases, have widely been adopted for this purpose, but can suffer from low activity, instability or limited substrate scope. Therefore, emerging oxygenating biocatalysts which offer an alternative will also be covered, as well as considering the ways in which these hydroxylation biotransformations can be applied in drug discovery and development, such as latestage functionalisation (LSF) and in biocatalytic cascades.

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Selective biocatalytic hydroxylation of unactivated methylene C–H bonds in cyclic alkyl substrates

Abstract: The monooxygenase, CYP101B1, selectively hydroxylates undistinct methylene C–H bonds in medium to large cycloalkyl rings and can generate oxabicycloundecanol derivatives.

Cited by 14 publications

References 34 publications

Mechanism-Guided Design and Discovery of Efficient Cytochrome P450-Derived C–H Amination Biocatalysts

Mechanism-Guided Design and Discovery of Efficient Cytochrome P450-Derived C–H Amination Biocatalysts

Harnessing P450 Enzyme for Biotechnology and Synthetic Biology

Oxygenating Biocatalysts for Hydroxyl Functionalisation in Drug Discovery and Development

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