Biocatalytic strategy for the construction of sp3-rich polycyclic compounds from directed evolution and computational modelling.

Catalysis with engineered enzymes has provided more efficient routes for the production of active pharmaceutical agents. However, the potential of biocatalysis to assist in early-stage drug discovery campaigns remains largely untapped. In this study, we have developed a biocatalytic strategy for the construction of sp3-rich polycyclic compounds via the intramolecular cyclopropanation of benzothiophenes and related heterocycles. Two carbene transferases with complementary regioisomer selectivity were evolved to catalyse the stereoselective cyclization of benzothiophene substrates bearing diazo ester groups at the C2 or C3 position of the heterocycle. The detailed mechanisms of these reactions were elucidated by a combination of crystallographic and computational analyses. Leveraging these insights, the substrate scope of one of the biocatalysts could be expanded to include previously unreactive substrates, highlighting the value of integrating evolutionary and rational strategies to develop enzymes for new-to-nature transformations. The molecular scaffolds accessed here feature a combination of three-dimensional and stereochemical complexity with 'rule-of-three' properties, which should make them highly valuable for fragment-based drug discovery campaigns.

Enantioselective Single and Dual α-C-H Bond Functionalization of Cyclic Amines via Enzymatic Carbene Transfer.

Cyclic amines are ubiquitous structural motifs found in pharmaceuticals and biologically active natural products, making methods for their elaboration via direct C-H functionalization of considerable synthetic value. Herein, we report the development of an iron-based biocatalytic strategy for enantioselective α-C-H functionalization of pyrrolidines and other saturated N-heterocycles via a carbene transfer reaction with diazoacetone. Currently unreported for organometallic catalysts, this transformation can be accomplished in high yields, high catalytic activity, and high stereoselectivity (up to 99:1 e.r. and 20,350 TON) using engineered variants of cytochrome P450 CYP119 from Sulfolobus solfataricus. This methodology was further extended to enable enantioselective α-C-H functionalization in the presence of ethyl diazoacetate as carbene donor (up to 96:4 e.r. and 18,270 TON), and the two strategies were combined to achieve a one-pot as well as a tandem dual C-H functionalization of a cyclic amine substrate with enzyme-controlled diastereo- and enantiodivergent selectivity. This biocatalytic approach is amenable to gram-scale synthesis and can be applied to drug scaffolds for late-stage C-H functionalization. This work provides an efficient and tunable method for direct asymmetric α-C-H functionalization of saturated N-heterocycles, which should offer new opportunities for the synthesis, discovery, and optimization of bioactive molecules.

Highly stereoselective and enantiodivergent synthesis of cyclopropylphosphonates with engineered carbene transferases.

Organophosphonate compounds have represented a rich source of biologically active compounds, including enzyme inhibitors, antibiotics, and antimalarial agents. Here, we report the development of a highly stereoselective strategy for olefin cyclopropanation in the presence of a phosphonyl diazo reagent as carbene precursor. In combination with a 'substrate walking' protein engineering strategy, two sets of efficient and enantiodivergent myoglobin-based biocatalysts were developed for the synthesis of both (1R,2S) and (1S,2R) enantiomeric forms of the desired cyclopropylphosphonate ester products. This methodology enables the efficient transformation of a broad range of vinylarene substrates at a preparative scale (i.e. gram scale) with up to 99% de and ee. Mechanistic studies provide insights into factors that contribute to make this reaction inherently more challenging than hemoprotein-catalyzed olefin cyclopropanation with ethyl diazoacetate investigated previously. This work expands the range of synthetically useful, enzyme-catalyzed transformations and paves the way to the development of metalloprotein catalysts for abiological carbene transfer reactions involving non-canonical carbene donor reagents.

Engineered and Artificial Metalloenzymes for Selective C-H Functionalization.

The direct functionalization of C-H bonds constitutes a powerful strategy to construct and diversify organic molecules. However, controlling the chemo- and site-selectivity of this transformation in particularly complex molecular settings represents a significant challenge. Metalloenzymes are ideal platforms for achieving catalyst-controlled selective C-H bond functionalization as their reactivities can be tuned by protein engineering and/or redesign of their cofactor environment. In this review, we highlight recent progress in the development of engineered and artificial metalloenzymes for C-H functionalization, with a focus on biocatalytic strategies for selective C-H oxyfunctionalization and halogenation as well as C-H amination and C-H carbene insertion via abiological nitrene and carbene transfer chemistries. Engineered heme- and non-heme iron dependent enzymes have emerged as promising scaffolds for executing these transformations with high chemo-, regio- and stereocontrol as well as tunable selectivity. These emerging systems and methodologies have expanded the toolbox of sustainable strategies for organic synthesis and created new opportunities for the generation of chiral building blocks, the late-stage C-H functionalization of complex molecules, and the total synthesis of natural products.

An Enzymatic Platform for the Highly Enantioselective and Stereodivergent Construction of Cyclopropyl-δ-lactones.

Abiological enzymes offers new opportunities for sustainable chemistry. Herein, we report the development of biological catalysts derived from sperm whale myoglobin that exploit a carbene transfer mechanism for the asymmetric synthesis of cyclopropane-fused-δ-lactones, which are key structural motifs found in many biologically active natural products. While hemin, wild-type myoglobin, and other hemoproteins are unable to catalyze this reaction, the myoglobin scaffold could be remodeled by protein engineering to permit the intramolecular cyclopropanation of a broad spectrum of homoallylic diazoacetate substrates in high yields and with up to 99 % enantiomeric excess. Via an alternate evolutionary trajectory, a stereodivergent biocatalyst was also obtained for affording mirror-image forms of the desired bicyclic products. In combination with whole-cell transformations, the myoglobin-based biocatalyst was used for the asymmetric construction of a cyclopropyl-δ-lactone scaffold at a gram scale, which could be further elaborated to furnish a variety of enantiopure trisubstituted cyclopropanes.

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

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.

Highly Stereoselective Synthesis of Fused Cyclopropane-γ-Lactams via Biocatalytic Iron-Catalyzed Intramolecular Cyclopropanation.

We report the development of an iron-based biocatalytic strategy for the asymmetric synthesis of fused cyclopropane-γ-lactams, which are key structural motifs found in synthetic drugs and bioactive natural products. Using a combination of mutational landscape and iterative site-saturation mutagenesis, sperm whale myoglobin was evolved into a biocatalyst capable of promoting the cyclization of a diverse range of allyl diazoacetamide substrates into the corresponding bicyclic lactams in high yields and with high enantioselectivity (up to 99% ee). These biocatalytic transformations can be performed in whole cells and could be leveraged to enable the efficient (chemo)enzymatic construction of chiral cyclopropane-γ-lactams as well as ß-cyclopropyl amines and cyclopropane-fused pyrrolidines, as valuable building blocks and synthons for medicinal chemistry and natural product synthesis.

Stereodivergent Intramolecular Cyclopropanation Enabled by Engineered Carbene Transferases.

We report the development of engineered myoglobin biocatalysts for executing asymmetric intramolecular cyclopropanations resulting in cyclopropane-fused γ-lactones, which are key motifs found in many bioactive molecules. Using this strategy, a broad range of allyl diazoacetate substrates were efficiently cyclized in high yields with up to 99% enantiomeric excess. Upon remodeling of the active site via protein engineering, myoglobin variants with stereodivergent selectivity were also obtained. In combination with whole-cell transformations, these biocatalysts enabled the gram-scale assembly of a key intermediate useful for the synthesis of the insecticide permethrin and other natural products. The enzymatically produced cyclopropyl-γ-lactones can be further elaborated to furnish a variety of enantiopure trisubstituted cyclopropanes. This work introduces a first example of biocatalytic intramolecular cyclopropanation and provides an attractive strategy for the stereodivergent preparation of fused cyclopropyl-γ-lactones of high value for medicinal chemistry and the synthesis of natural products.

Hydroxylation of anilides by engineered cytochrome P450BM3.

Biocatalytic direct monohydroxylation of anilides has been achieved on preparative scale using mutant cytochrome P450BM3 enzymes. Representative mono- and disubstituted N-trifluoromethanesulfonyl anilides are shown to be converted in most cases to the corresponding 4-hydroxy derivatives, with substituent hydroxylation also occurring in two cases. By mutation variation, it is possible to achieve selective hydroxylation of either ring- or side-chain sites.

Drug Oxidation by Cytochrome P450BM3 : Metabolite Synthesis and Discovering New P450 Reaction Types.

There is intense interest in late-stage catalytic C-H bond functionalization as an integral part of synthesis. Effective catalysts must have a broad substrate range and tolerate diverse functional groups. Drug molecules provide a good test of these attributes of a catalyst. A library of P450BM3 mutants developed from four base mutants with high activity for hydrocarbon oxidation produced human metabolites of a panel of drugs that included neutral (chlorzoxazone, testosterone), cationic (amitriptyline, lidocaine) and anionic (diclofenac, naproxen) compounds. No single mutant was active for all the tested drugs but multiple variants in the library showed high activity with each compound. The high conversions enabled full product characterization that led to the discovery of the new P450 reaction type of oxidative decarboxylation of an α-hydroxy carboxylic acid and the formation a protected imine from an amine, offering a novel route to α-functionalization of amines. The substrate range and varied product profiles suggest that this library of enzymes is a good basis for developing late-stage C-H activation catalysts.

A new method for analysis of the toxicity of organophosphorus pesticide, dimethoate on rotifer based on response surface methodology.

This study analyzed the toxicity of organophosphorus pesticide, dimethoate on freshwater rotifer Brachionus calyciflorus, using swimming angular and linear speed alteration as the sub-lethal endpoints. Response surface methodology (RSM) was applied in experimental design and data analysis to consider two related factors: toxic concentration, exposure time and their interaction. In general, inhibition effect of the pesticide on rotifer swimming was observed clearly at any given toxicant concentration. The highest inhibition rates in angular and linear speed were obtained in the shortest exposure time (11.36 min) and the highest dimethoate concentration (1.85 mg L(-1)). The RSM used for the analysis of treatment combinations showed that a cubic polynomial regression model was in good agreement with experimental results, with R(2)=0.992 and 0.9997, for swimming angular speed inhibition rate and linear speed inhibition rate (p<0.01, F-test, respectively). 3D reference surface plots and contour plots showed that the toxic effect was influenced not only by dimethoate concentration, but also by the exposure time. A time-step effect was observed clearly. Thus, the pesticide dimethoate had toxic stress on the swimming behavior of rotifers.

Assessment the toxic effects of dimethoate to rotifer using swimming behavior.

The toxic effects of the common organophosphorus pesticide dimethoate on freshwater zooplankton Brachionus calyciflorus (rotifer) were tested. Because of the advantages of behavioral response in environmental monitoring, swimming behavior was used as the endpoint in this research. After exposure 6 h at five dimethoate concentrations (0.18, 0.53, 0.88, 1.23 and 1.59 mg·L(-1)), the pesticide disrupted the balance in rotifer swimming direction and caused an obvious direction preference. It also inhibited significantly the swimming angular and linear speed. Our results showed that dimethoate has a sublethal toxic effect on this aquatic invertebrate.