R.B. Leveson-Gower

We describe an enantioselective C4-dearomatization of methoxypyridine derivatives for the preparation of functionalised enantioenriched δ-lactams using chiral copper catalysis. Experimental ¹³C kinetic isotope effects and density functional theory calculations shed light on the reaction mechanism and the origin of enantioselectivity.

Abstract

Nitrogen-bearing rings are common features in the molecular structures of modern drugs, with chiral δ-lactams being an important subclass due to their known pharmacological properties. Catalytic dearomatization of preactivated pyridinium ion derivatives emerged as a powerful method for the rapid construction of chiral N-heterocycles. However, direct catalytic dearomatization of simple pyridine derivatives are scarce and methodologies yielding chiral δ-lactams are yet to be developed. Herein, we describe an enantioselective C4-dearomatization of methoxypyridine derivatives for the preparation of functionalised enantioenriched δ-lactams using chiral copper catalysis. Experimental ¹³C kinetic isotope effects and density functional theory calculations shed light on the reaction mechanism and the origin of enantioselectivity.

Nature Catalysis, Published online: 08 December 2022; doi:10.1038/s41929-022-00879-z

Iridium catalysis can be used to achieve the challenging Z-retentive asymmetric allylic substitution reaction by trapping thermodynamically less stable anti-π-allyliridium intermediates. Now the isolation and characterization of these complexes is reported, providing hitherto elusive detailed mechanistic insights into this reaction.

Nature, Published online: 13 December 2022; doi:10.1038/d41586-022-04440-7

Researchers at the US National Ignition Facility created a reaction that made more energy than they put in.

The enantioselective addition of water across unactivated alkenes is a much sought-after chemical transformation and a major challenge in catalysis. Now a promiscuous engineered fatty acid hydratase produces chiral alcohols with high enantioselectivity, also on a preparative scale, using simple alkenes and water as reactants.

Abstract

Enantioselective synthesis of chiral alcohols through asymmetric addition of water across an unactivated alkene is a highly sought-after transformation and a big challenge in catalysis. Herein we report the identification and directed evolution of a fatty acid hydratase from Marinitoga hydrogenitolerans for the highly enantioselective hydration of styrenes to yield chiral 1-arylethanols. While directed evolution for styrene hydration was performed in the presence of heptanoic acid to mimic fatty acid binding, the engineered enzyme displayed remarkable asymmetric styrene hydration activity in the absence of the small molecule activator. The evolved styrene hydratase provided access to chiral alcohols from simple alkenes and water with high enantioselectivity (>99 : 1 e.r.) and could be applied on a preparative scale.

Nature Chemical Biology, Published online: 12 December 2022; doi:10.1038/s41589-022-01206-0

The specificity constant of a promiscuous enzyme was raised by over 1,000-fold by using computational protein design to place a substrate recruitment domain adjacent to the enzyme active site.

The stereoselective Michael addition of pyruvate to β-nitrostyrenes catalyzed by NahE, a type 1 aldolase, is reported. β-Aryl-γ-nitrobutyric acids can be isolated after oxidative decarboxylation in high yields on a preparative scale, providing access to precursors of γ-aminobutyric acid (GABA) analogues of demonstrated pharmacological activity.

Abstract

Michael addition reactions are highly useful in organic synthesis and are commonly accomplished using organocatalysts. However, the corresponding biocatalytic Michael additions are rare, typically lack synthetically useful substrate scope, and suffer from low stereoselectivity. Herein we report a biocatalytic nitro-Michael addition, catalyzed by NahE, that proceeds with low catalyst loading at room temperature in moderate to excellent enantioselectivity and high yields. A series of β-nitrostyrenes reacted with pyruvate in the presence of NahE to give, after oxidative decarboxylation, β-aryl-γ-nitrobutyric acids in up to 99 % yield without need for chromatography, providing a simple preparative-scale route to chiral GABA analogues. This reaction represents the first example of an aldolase displaying promiscuous Michaelase activity and opens the use of nitroalkenes in place of aldehydes as substrates for aldolases.

The conversion of CO2 by enzymes such as carbonic anhydrases or carboxylases plays a crucial role in many biological processes. However, methods to study the conversion of CO2 at the active site of enzymes in situ are still limited. Here, we used Fourier-transform infrared (FTIR) spectroscopy to study the interaction of CO2 with crotonyl-CoA carboxylase/reductase from Kitasaospora setae (KsCcr), one of the fastest CO2-fixing enzymes in nature. Our studies reveal that the enzyme possesses a so far unknown metal-independent carbonic anhydrase activity. Molecular dynamics (MD) simulations explain why substrate binding inhibits anhydrase activity, and mutations of active site residues of KsCcr suggest that an ‘activated’ water molecule, coordinated by a histidine and glutamate residue, forms the hydroxyl anion that attacks the CO2 molecule. Altogether, we demonstrate how in situ FTIR spectroscopy combined with MD simulations provides new means to investigate the interaction of different proteins with CO2, providing a simple, yet powerful approach to atomistic reaction mechanisms including CO2 hydration and enzymatic (de-)hydration reactions.

A photoenzymatic strategy for radical-mediated stereoselective hydroalkylation with diazo compounds has been developed. By this method, a series of γ-chiral carbonyl compounds were synthesized in high yields and stereoselectivities.

Abstract

Carbene insertion reactions initiated with diazo compounds have been widely used to develop unnatural enzymatic reactions. However, alternative functionalization of diazo compounds in enzymatic processes has been unexploited. Herein, we describe a photoenzymatic strategy for radical-mediated stereoselective hydroalkylation with diazo compounds. This method generates carbon-centered radicals through an ene reductase catalyzed photoinduced electron transfer process from diazo compounds, enabling the synthesis of γ-stereogenic carbonyl compounds in good yields and stereoselectivities. This study further expands the possible reaction patterns in photo-biocatalysis and offers a new approach to solving the selectivity challenges of radical-mediated reactions.

Nature Chemical Biology, Published online: 05 December 2022; doi:10.1038/s41589-022-01179-0

Anaplerotic reactions constantly refill metabolic networks with essential intermediates. This concept was adapted to enable a 54-step in vitro biosynthesis of the macrolide backbone of the antibiotic erythromycin from CO2.

Nature Communications, Published online: 03 December 2022; doi:10.1038/s41467-022-35228-y

Fast screening of enzymes is key for directed evolution of industrial biocatalysts. Here, the authors report a simple, high-throughput, and low-equipment-dependent growth selection system for engineering three enzymes for synthesis of chiral amines.

Visible light absorbing iridium(III) complexes have been widely employed as photocatalysts in organic synthesis. Their catalytic reactivity and selectivity are generally optimized by modifying the structures of coordinated ligands to obtain desired photophysical properties. Artificial metalloenzymes (ArMs) can combine the unique features of both metal complexes and enzymes by incorporating a cofactor within a protein scaffold, which offers another strategy to improve the performance of metal catalysts. Herein, we describe a panel of Ir(III)-ArMs constructed by covalently embedding iridium(III) polypyridyl complexes into a prolyl oligopeptidase scaffold. A series of spectroscopic methods were used to examine how properties of the resulting ArMs are influenced by structural variation of the cyclometalated ligands and the protein scaffold. Visible light photocatalysis by these hybrid catalysts was also examined, leading to the finding that they catalyze inter/intra-molecular [2+2] photocycloaddition in aqueous solution and indicating that they can serve as new bio-photocatalysts for further exploration.

We repurposed the metal-binding site of a cupin superfamily protein into the 2-his-1-carboxylate facial triad, which is one of the common motifs in natural non-heme enzymes, to construct artificial metalloenzymes that can catalyze new-to-nature reactions. Cu2+-H52A/H58E variant catalyzed the stereoselective Michael addition reaction and was found to bear a flexible metal-binding site in the high-resolution crystal structure. Furthermore, the H52A/H58E/F104W mutant accommodated a water molecule, which was supported by Glu58 and Trp104 residues via hydrogen bonding, presumably leading to high stereoselectivity. Thus, the 2-his-1-carboxylate facial triad was confirmed to be a versatile and promising metal-binding motif for abiological and canonical biological reactions.

Nature Catalysis, Published online: 28 November 2022; doi:10.1038/s41929-022-00875-3

Carbonyl catalysis is mainly limited to strongly activated primary amines. Now, a chiral bifunctional pyridoxal organocatalyst is developed that enables the activation of the inert α C(sp3)–H bond of NH2-unprotected benzylamines affording chiral β-aminoalcohols with high diastereo- and enantioselectivities.

The design of artificial enzymes has emerged as a promising tool for the generation of potent biocatalysts able to promote new-to-nature reactions. This review aims to give a general overview of suitable protein scaffolds, that could be functionalized with an artificial moiety to develop versatile artificial catalysts.

Abstract

The design of artificial enzymes has emerged as a promising tool for the generation of potent biocatalysts able to promote new-to-nature reactions with improved catalytic performances, providing a powerful platform for wide-ranging applications and a better understanding of protein functions and structures. The selection of an appropriate protein scaffold plays a key role in the design process. This review aims to give a general overview of the most common protein scaffolds that can be exploited for the generation of artificial enzymes. Several examples are discussed and categorized according to the strategy used for the design of the artificial biocatalyst, namely the functionalization of natural enzymes, the creation of a new catalytic site in a protein scaffold bearing a wide hydrophobic pocket and de novo protein design. The review is concluded by a comparison of these different methods and by our perspective on the topic.

Nature Catalysis, Published online: 17 November 2022; doi:10.1038/s41929-022-00873-5

Artificial enzymes capable of catalysing significant transformations are highly desired but usually suffer from limitations in structural design and poor efficiency. Now, a monolayered metal–organic framework is reported as an editable biomimetic platform to achieve exceptional artificial photosynthesis performance.

Tertiary nitroalkanes and the corresponding α-tertiary amines represent important motifs in bioactive molecules and natural prod-ucts. The C-alkylation of secondary nitroalkanes with electrophiles is a straightforward strategy for constructing tertiary nitroal-kanes, however, controlling the stereoselectivity of this type of reaction remains challenging. Here we report a highly chemo- and stereoselective C-alkylation of nitroalkanes with alkyl halides catalyzed by an engineered flavin-dependent ‘ene’-reductase (ERED). Directed evolution of the old yellow enzyme from Geobacillus kaustophilus provided a triple mutant, GkOYE-G7, capable of synthesizing tertiary nitroalkanes with high yield and enantioselectivity. Mechanistic studies indicate that the excitation of an enzyme-templated charge-transfer complex formed between the substrates and cofactor is responsible for radical initiation. Moreover, a single-enzyme two-mechanism cascade reaction was developed to prepare tertiary nitroalkanes from simple nitroal-kenes, highlighting the potential to use one enzyme for two mechanistically distinct reactions.

Nature Chemistry, Published online: 14 November 2022; doi:10.1038/s41557-022-01083-z

Expanding the biocatalysis toolbox for C–N bond formation is of great value. Now, a biocatalytic amination strategy using a new-to-nature mechanism involving nitrogen-centred radicals has been developed. The transformations are enabled by synergistic photoenzymatic catalysis, providing intra- and intermolecular hydroamination products with high yields and levels of enantioselectivity.

The Prelog rule in l-threonine aldolase holds that when the C_α anion of PLP-Gly attacks the carbonyl carbon atom of the aldehyde from the re-face, the (2S,3S)-configured product is formed, whereas attack from the si-face forms the (2S,3R)-configured product. Guided by this rule, mutants of LTA with improved diastereoselectivity of 99.2 % _syn and 97.4 % _anti were obtained.

Abstract

l-threonine aldolase (LTA) catalyzes C−C bond synthesis with moderate diastereoselectivity. In this study, with LTA from Cellulosilyticum sp (CpLTA) as an object, a mutability landscape was first constructed by performing saturation mutagenesis at substrate access tunnel amino acids. The combinatorial active-site saturation test/iterative saturation mutation (CAST/ISM) strategy was then used to tune diastereoselectivity. As a result, the diastereoselectivity of mutant H305L/Y8H/V143R was improved from 37.2 % _syn to 99.4 % _syn . Furthermore, the diastereoselectivity of mutant H305Y/Y8I/W307E was inverted to 97.2 % _anti . Based on insight provided by molecular dynamics simulations and coevolution analysis, the Prelog rule was employed to illustrate the diastereoselectivity regulation mechanism of LTA, holding that the asymmetric formation of the C−C bond was caused by electrons attacking the carbonyl carbon atom of the substrate aldehyde from the re or si face. The study would be useful to expand LTA applications and guide engineering of other C−C bond-forming enzymes.

An in vivo selection strategy is presented, in which bacteria addicted to non-canonical amino acids (ncAAs) are complemented by enzymes that can yield these building blocks from synthetic precursors. As growth rates under selective conditions correlate with enzyme activities, serial passaging elicited better biocatalysts from populations harboring enzyme libraries. The platform was used to improve the activity of carbamoylases for ncAA-precursors.

Abstract

In vivo selections are powerful tools for the directed evolution of enzymes. However, the need to link enzymatic activity to cellular survival makes selections for enzymes that do not fulfill a metabolic function challenging. Here, we present an in vivo selection strategy that leverages recoded organisms addicted to non-canonical amino acids (ncAAs) to evolve biocatalysts that can provide these building blocks from synthetic precursors. We exemplify our platform by engineering carbamoylases that display catalytic efficiencies more than five orders of magnitude higher than those observed for the wild-type enzyme for ncAA-precursors. As growth rates of bacteria under selective conditions correlate with enzymatic activities, we were able to elicit improved variants from populations by performing serial passaging. By requiring minimal human intervention and no specialized equipment, we surmise that our strategy will become a versatile tool for the in vivo directed evolution of diverse biocatalysts.

We report in vivo biocatalytic cascade reactions comprising a combination of canonical enzyme-catalysed reactions with an artificial-enzyme-catalysed new-to-nature reaction. The artificial enzyme contains a genetically encoded unnatural catalytic residue, which catalyses the formation of a hydrazone product from biosynthetically produced benzaldehydes in E. coli.

Abstract

Artificial enzymes utilizing the genetically encoded non-proteinogenic amino acid p-aminophenylalanine (pAF) as a catalytic residue are able to react with carbonyl compounds through an iminium ion mechanism to promote reactions that have no equivalent in nature. Herein, we report an in vivo biocatalytic cascade that is augmented with such an artificial enzyme-catalysed new-to-nature reaction. The artificial enzyme in this study is a pAF-containing evolved variant of the lactococcal multidrug-resistance regulator, designated LmrR_V15pAF_RMH, which efficiently converts benzaldehyde derivatives produced in vivo into the corresponding hydrazone products inside E. coli cells. These in vivo biocatalytic cascades comprising an artificial-enzyme-catalysed reaction are an important step towards achieving a hybrid metabolism.