R.B. Leveson-Gower

Structural diversification of lead molecules is a key component of drug discovery to explore close-in chemical space. Late stage functionalizations (LSFs) are versatile methodologies capable of installing functional handles on richly decorated intermediates to deliver numerous diverse products in a single reaction. Predicting the regioselectivity of LSF is still an open challenge in the field. Numerous efforts from chemoinformatics and machine learning (ML) groups have made significant strides in this area. However, it is arduous to isolate and characterize the multitude of LSF products generated, limiting available data and hindering pure ML approaches. We report the development of an approach that combines message-passing neural network and an 13C NMR-based transfer learning to predict the atom-wise probabilities of functionalization. We validated our model retrospectively and with a series of prospective experiments, showing that it accurately predicts the outcomes of Minisci-type and P450 transformations, outperforming state-of-the-art Fukui-based reactivity indices.

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.

Nature Chemistry, Published online: 22 December 2022; doi:10.1038/s41557-022-01082-0

Macrocyclic peptides can be genetically encoded and synthesized in cells; however, the programmable diversity is limited. Now, macrocycles containing two non-canonical amino acids have been genetically encoded and synthesized in codon-reassigned Syn61Δ3 cells. Incorporating diverse hydroxy acids in Syn61Δ3 cells enables the synthesis of non-natural depsipeptides containing either one or two ester bonds.

Protoglobins were engineered to catalyze stereodivergent cyclopropanation to afford a variety of trifluoromethyl-substituted cyclopropanes using trifluorodiazoethane as the trifluoromethyl-carbene precursor.

Abstract

Trifluoromethyl-substituted cyclopropanes (CF₃-CPAs) constitute an important class of compounds for drug discovery. While several methods have been developed for synthesis of trans-CF₃-CPAs, stereoselective production of corresponding cis-diastereomers remains a formidable challenge. We report a biocatalyst for diastereo- and enantio-selective synthesis of cis-CF₃-CPAs with activity on a variety of alkenes. We found that an engineered protoglobin from Aeropyrnum pernix (ApePgb) can catalyze this unusual reaction at preparative scale with low-to-excellent yield (6–55 %) and enantioselectivity (17–99 % ee), depending on the substrate. Computational studies revealed that the steric environment in the active site of the protoglobin forced iron-carbenoid and substrates to adopt a pro-cis near-attack conformation. This work demonstrates the capability of enzyme catalysts to tackle challenging chemistry problems and provides a powerful means to expand the structural diversity of CF₃-CPAs for drug discovery.

Nature Communications, Published online: 19 December 2022; doi:10.1038/s41467-022-35468-y

Chiral heterocyclic compounds are privileged structures in medicinal chemistry. Here, the authors report an in silico strategy for the enzymatic synthesis of pharmaceutically significant chiral N- and O-heterocycles via Baldwin cyclization of hydroxy- and amino-substituted epoxides and oxetanes using epoxide hydrolase mutants.

A new cyclisation approach for peptides, derived from in vitro translation, is developed that is highly selective for N-terminal cysteines. This approach allows other cysteine residues to be exploited for further peptide diversification. A model peptide showed a large impact from this cyclisation on binding and biological activity, and molecular dynamics was used to illustrate how this cyclisation change can influence peptide conformation.

Abstract

Macrocyclisation provides a means of stabilising the conformation of peptides, often resulting in improved stability, selectivity, affinity, and cell permeability. In this work, a new approach to peptide macrocyclisation is reported, using a cyanobenzothiazole-containing amino acid that can be incorporated into peptides by both in vitro translation and solid phase peptide synthesis, meaning it should be applicable to peptide discovery by mRNA display. This cyclisation proceeds rapidly, with minimal by-products, is selective over other amino acids including non N-terminal cysteines, and is compatible with further peptide elaboration exploiting such an additional cysteine in bicyclisation and derivatisation reactions. Molecular dynamics simulations show that the new cyclisation group is likely to influence the peptide conformation as compared to previous thioether-based approaches, through rigidity and intramolecular aromatic interactions, illustrating their complementarity.

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.