R.B. Leveson-Gower

Nature, Published online: 22 June 2022; doi:10.1038/s41586-022-04862-3

Global ocean microbiome survey reveals the bacterial family ‘Candidatus Eudoremicrobiaceae’, which includes some of the most biosynthetically diverse microorganisms in the ocean environment.

A PluriZyme with transaminase and esterase activities was constructed by combining computational and laboratory design methods. The enzyme can perform cascade reactions in a single protein scaffold. Experimental evidence is presented for the catalytic efficiency of both the native transaminase and the artificial esterase active site and their synergistic action to transform β-keto esters into enantiopure β-amino acids.

Abstract

Engineering dual-function single polypeptide catalysts with two abiotic or biotic catalytic entities (or combinations of both) supporting cascade reactions is becoming an important area of enzyme engineering and catalysis. Herein we present the development of a PluriZyme, TR₂E₂, with efficient native transaminase (k _cat: 69.49±1.77 min⁻¹) and artificial esterase (k _cat: 3908–0.41 min⁻¹) activities integrated into a single scaffold, and evaluate its utility in a cascade reaction. TR₂E₂ (pH_opt: 8.0–9.5; T _opt: 60–65 °C) efficiently converts methyl 3-oxo-4-(2,4,5-trifluorophenyl)butanoate into 3-(R)-amino-4-(2,4,5-trifluorophenyl)butanoic acid, a crucial intermediate for the synthesis of antidiabetic drugs. The reaction proceeds through the conversion of the β-keto ester into the β-keto acid at the hydrolytic site and subsequently into the β-amino acid (e.e. >99 %) at the transaminase site. The catalytic power of the TR₂E₂ PluriZyme was proven with a set of β-keto esters, demonstrating the potential of such designs to address bioinspired cascade reactions.

Spatial control over chemical reactions is a ubiquitous feature shared by all living organisms. In the present article, we describe the design of a proteinaceous, synthetic capsid based on the well-described bacterial nanocompartment encapsulin. Our engineered virus-like particle carries a dual functional artificial metalloenzyme (ArM) as non-native guest protein. This ArM, created from a fusion protein of HaloTag and monomeric rhizavidin, serves as catalyst for a fully bioorthogonal, linear, two-step reaction cascade. A ruthenium-catalyzed alloc deprotection is followed by a gold-catalyzed, ring-closing hydroamination reaction leading to indoles and phenanthridines with up to 67 % overall yield in aqueous solutions.

Nature Communications, Published online: 22 June 2022; doi:10.1038/s41467-022-31293-5

The activation of aziridines is typically achieved via reaction with strong Lewis acids or transition metals. Here, the authors report that cooperative Se ∙ ∙∙O and Se ∙ ∙∙N noncovalent interactions can activate sulfonyl-protected aziridines, which enables their use in cycloaddition reactions with nonactivated alkenes.

Nature Catalysis, Published online: 16 June 2022; doi:10.1038/s41929-022-00800-8

Flavin-dependent halogenases catalyse the challenging regioselective halogenation of aromatic compounds, but display low efficiency. Now, a tryptophan-halogenase with multiple catalytic improvements is obtained by engineering the intermediate transfer tunnel connecting the enzyme´s two active sites.

Come together: Asymmetric aminocatalysis plays a pivotal role on the development in stereoselective transformations, synergistically combined with organo-, metal, photoredox and electrocatalysis. In this scenario, the HOMO-raising and LUMO-lowering activation of carbonyl compounds open new gateways to complex architectures and new sustainable reactivities. Last but not least, this research area will keep driving the development of the whole organocatalysis, toward its synergistic combination with other activation strategies.

Abstract

Synergistic catalysis offers the unique possibility of simultaneous activation of both the nucleophile and the electrophile in a reaction. A requirement for this strategy is the stability of the active species towards the reaction conditions and the two concerted catalytic cycles. Since the beginning of the century, aminocatalysis has been established as a platform for the stereoselective activation of carbonyl compounds through HOMO-raising or LUMO-lowering. The burgeoning era of aminocatalysis has been driven by a deep understanding of these activation and stereoinduction modes, thanks to the introduction of versatile and privileged chiral amines. The aim of this review is to cover recent developments in synergistic strategies involving aminocatalysis in combination with organo-, metal-, photo-, and electro-catalysis, focusing on the evolution of privileged aminocatalysts architectures.

A visible light mediated Arbuzov type reaction of thiophenols and phosphites to access phosphorothioates under mild metal-free conditions was demonstrated.

Abstract

We hereby disclose, a visible light mediated addition of sulfenyl radicals to trialkyl phosphites to access functionalized phosphorothioates. The use of cheap and readily available Eosin Y as a photocatalyst under mild energy efficient conditions bypassing the use of external oxidants forms the chief highlight of the work. The protocol is scalable and mechanistic studies indicate that the reaction proceeds through an ionic-Arbuzov like pathway from phosphoranyl radicals.

Diastereomeric intermediates can be separated by ion mobility mass spectrometry. Monitoring of the kinetics of the intermediates allows the prediction of the overall reaction enantioselectivity. The experiments can be performed in a small scale and do not require an isolation of the products.

Abstract

Enantioselective reactions are at the core of chemical synthesis. Their development mostly relies on prior knowledge, laborious product analysis and post-rationalization by theoretical methods. Here, we introduce a simple and fast method to determine enantioselectivities based on mass spectrometry. The method is based on ion mobility separation of diastereomeric intermediates, formed from a chiral catalyst and prochiral reactants, and delayed reactant labeling experiments to link the mass spectra with the reaction kinetics in solution. The data provide rate constants along the reaction paths for the individual diastereomeric intermediates, revealing the origins of enantioselectivity. Using the derived kinetics, the enantioselectivity of the overall reaction can be predicted. Hence, this method can offer a rapid discovery and optimization of enantioselective reactions in the future. We illustrate the method for the addition of cyclopentadiene (CP) to an α,β-unsaturated aldehyde catalyzed by a diarylprolinol silyl ether.

A small artificial hydrolase that mimics the active centre of chymotrypsin and N-terminal hydrolases is able to perform, for the first time, the transesterification of ethyl acetate with methanol under neutral conditions and at room temperature. This artificial enzyme also catalysed the transesterification of important neurotransmitter acetylcholine into methyl acetate and choline.

Abstract

The synthesis of small molecules able to mimic the active site of hydrolytic enzymes has been largely pursued in recent decades. The high reaction rates and specificity shown by natural hydrolases present an attractive target, and yet the preparation of suitable small-molecule mimics remains challenging, requiring activated substrates to achieve productive outcomes. Here we present small synthetic artificial enzymes which mimic the catalytic site and the oxyanion hole of chymotrypsin and N-terminal hydrolases and are able to perform, for the first time, the transesterification of a non-activated ester such as ethyl acetate with methanol under mild and neutral reaction conditions.

Isothiouronyl radical cations have been identified as a new class of radical catalysts for the covalent activation of vinylcyclopropanes. Their generation by photoredox catalysis from N,N,N,N-tetrasubstituted thioureas provides a modular, versatile and practical platform to achieve covalent radical catalysis.

Abstract

Thiyl radicals offer unique catalytic patterns for the direct covalent activation of alkenes. However, important limitations in terms of structural diversity and handling have hampered the routine use of thiyl radicals in covalent radical catalysis. Herein, we report a new class of cationic sulfur-centered radicals to achieve covalent radical catalysis. Their generation from highly modular thioureas by photoredox catalysis make their utilization very simple and reliable. The synthetic potential and the versatility of the catalytic system were finally evaluated in a (3+2)-radical cascade between vinylcyclopropanes and olefins.

Herein we describe a method for combining supramolecular catalysis with imininum-based organocatalysis in the Diels–Alder cycloaddition reaction. Notably, both cage and L-proline are required for the reaction to occur, implying that encap-sulation of the substrates and co-catalyst are necessary for the reaction to occur. We explore the substrate scope for a va-riety of E-cinnamaldehydes and dienes. Finally, we probe the supramolecular assembly processes responsible for the observed catalysis using NMR spectroscopic methods, determining that the reaction is negative order in the cinnamalde-hyde substrate and experiences product inhibition.

Nature, Published online: 01 June 2022; doi:10.1038/s41586-022-04456-z

Recent progress in computational enzyme design, active site engineering and directed evolution are reviewed, highlighting methodological innovations needed to deliver improved designer biocatalysts.

Nature, Published online: 01 June 2022; doi:10.1038/s41586-022-04773-3

Chimeric triterpene synthases are identified that catalyse non-squalene-dependent triterpene biosynthesis.

Bio-Chem collaboration: Escherichia coli fermentation and visible-light photocatalysis were combined in a complementary way for the first time, to realize the scaffold assembly in a “one-pot” fashion and the subsequent diversifications in a “one-step” manner, respectively, enabling the production of six sesquiterpenoids.

Abstract

The diversification of natural products to expand biologically relevant chemical space for drug discovery can be achieved by combining complementary bioprocessing and chemical transformations. Herein, genetically engineered Escherichia coli fermentation to produce amorphadiene and valencene was combined with metal-free photocatalysis transformations to further access nootkatone, cis-nootkatol and two hydration derivatives. In fermentation, using a closed, anaerobic condition avoided the use of organic overlay, increased the productivity, and simplified the work-up process. Metal-free photocatalysis hydration and allylic C−H oxidation were designed and implemented to make the whole process greener. It was shown that the anti-Markovnikov selectivity of photocatalyzed alkene hydration could be reversed by stereo-electronic and steric effects existing in complex natural products. The combination of bioprocessing and photocatalysis may provide an efficient and greener way to expand the chemical space for pharmaceutical, flavor and fragrance industry.

Styrene monooxygenase catalyzed enantioselective anti-Markovnikov hydration of aryl alkenes, rather than epoxidation, was made possible by simply removing oxygen from the reaction mixture. An acid–base mechanism with a carbanion-like intermediate enabled the reaction with excellent anti-Markovnikov regioselectivity.

Abstract

The addition of water to alkenes is an important method for the synthesis of alcohols, but the regioselectivity of acid-catalyzed hydration of terminal alkenes yields secondary alcohols according to Markovnikov's rule, making it difficult to obtain primary alcohols. Here we report a styrene monooxygenase that catalyzes the anti-Markovnikov hydration of the terminal aryl alkenes under anaerobic conditions. This hydration provides primary alcohols in good yields (up to 100 %), excellent anti-Markovnikov regioselectivity (>99 : 1), and good enantiomeric purity (60–83 % ee). Residues Asn46, Asp100, and Asn309 are essential for catalysis suggesting an acid–base mechanism with a carbanion-like intermediate that could account for the anti-Markovnikov regioselectivity. Our work reveals a new enzymatic tool with unusual regioselectivity based on the promiscuous catalytic activity of a monooxygenase.

Asymmetric catalysis has witnessed paramount lessons from terpene cyclase enzymology such as the ability to control dynamic carbocations or cationic cyclization cascades. In general, these cascades are stereodivergent and thus rely on the terpene’s double-bond geometry. In this work, we illuminate how the dynamic supramolecular framework of squalene-hopene cyclases (SHCs) can be tailored to break with this paradigm. Creating a locally electron-enriched confined active site, we enabled the stereoconvergent cationic cyclization of a cis/trans terpene mixture into one isomer. Our results suggest that a priorly unknown active site “memory” effect of the SHC aids this intricate transformation. Based on these findings, we employed synergistic active site and tunnel engineering to generate a most efficient (–)-ambroxide cyclase. Broad computational investigations evidently explain how the introduced mutations work in concert to improve substrate acquisition, flow and chaperoning. Nonetheless, kinetics disclosed a substrate-induced downregulation of the membrane-bound SHC as the major turnover limitation in vivo. Merging these new insights with the improved and stereoconvergent catalysis of the enzyme, we applied a feeding strategy to exceed 106 TTN with the SHC.

An asymmetric oxidative cross-coupling strategy that combines the non-natural catalytic activity of lipase with organic electrosynthesis was developed. This unprecedented protocol demonstrates that hydrolase catalysis is compatible with electrosynthesis.

Abstract

We describe the enantioselective oxidative cross-coupling of secondary amines with ketones by combining the non-natural catalytic activity of lipase with electrosynthesis. Various 2,2-disubstituted 3-carbonyl indoles with a stereogenic quaternary carbon center were synthesized from 2-substituted indoles in yields up to 78 % with good enantio- and diastereoselectivities (up to 96 : 4 e.r. and >20 : 1 d.r.). This unprecedented protocol demonstrated that hydrolase catalysis is compatible with electrosynthesis, and the reaction can be carried out in organic solvents with a broad substrate scope and good stereoselectivity. This work provides insights into enzymatic electrosynthesis.