R.B. Leveson-Gower

In this work, we report a computationally driven approach to access enantiodivergent enzymatic carbene N–H bond insertions catalyzed by P411 enzyme variants. Computational modeling was employed to guide engineering efforts to control the accessible conformations of a key lactone-carbene (LAC) intermediate in the enzyme active site by installing a new H-bond anchoring point. By combining MD simulations and protein engineering, a reversed (R-selective) P411 enzyme variant, L5_FL-B3, was obtained in a single round of semi-rational directed evolution. L5_FL-B3 accepts a broad scope of amine substrates with excellent yields (up to >99%), high efficiency (up to 12,300 TTN) and good enantiocontrol (up to 7:93 er), which complements the previously engineered S-selective P411-L7_LF variant.

Nature Catalysis, Published online: 11 August 2022; doi:10.1038/s41929-022-00821-3

Enzymes for poly(ethylene terephthalate) (PET) deconstruction are of interest for plastics recycling, but reports on their directed evolution are missing. Now, an automated, high-throughput directed evolution platform is described, affording HotPETase that effectively achieves depolymerization above the glass transition temperature of PET.

Nature, Published online: 03 August 2022; doi:10.1038/s41586-022-05022-3

Biochemical and lipidomic analyses identify an anti-ferroptotic function of vitamin K and reveal ferroptosis suppressor protein 1 (FSP1) as the enzyme mediating warfarin-resistant vitamin K reduction in the canonical vitamin K cycle.

Nature, Published online: 11 August 2022; doi:10.1038/s41586-022-05167-1

An Asymmetric sp³–sp³ Cross-Electrophile Coupling Using ‘Ene’-Reductases

YfeX, naturally a peroxidase, has great potential for the development of new carbene transferases. WT YfeX catalyzes N−H insertion (including aliphatic and secondary amines) in high yield, cyclopropanation, and most excitingly, Si−H insertion of dimethylphenylsilane. QM/MM calculations reveal details of the mechanism of the unusual Si−H insertion reaction.

Abstract

Carbene transfer biocatalysis has evolved from basic science to an area with vast potential for the development of new industrial processes. In this study, we show that YfeX, naturally a peroxidase, has great potential for the development of new carbene transferases, due to its high intrinsic reactivity, especially for the N−H insertion reaction of aromatic and aliphatic primary and secondary amines. YfeX shows high stability against organic solvents (methanol and DMSO), greatly improving turnover of hydrophobic substrates. Interestingly, in styrene cyclopropanation, WT YfeX naturally shows high enantioselectivity, generating the trans product with 87 % selectivity for the (R,R) enantiomer. WT YfeX also catalyzes the Si−H insertion efficiently. Steric effects in the active site were further explored using the R232A variant. Quantum Mechanics/Molecular Mechanics (QM/MM) calculations reveal details on the mechanism of Si−H insertion. YfeX, and potentially other peroxidases, are exciting new targets for the development of improved carbene transferases.

We present a de novo discovery of an efficient catalyst of the Morita–Baylis–Hillman (MBH) reaction by searching chemical space for molecules that lower the estimated barrier of the rate determining step using a genetic algorithm (GA) starting from randomly selected tertiary amines. We performed five independent GA searches that resulted in 448 unique molecules, for which we were able to locate 435 true transitions states at semiempirical level of theory. The predicted activation energies of all 435 molecules where all lower than that of DABCO, which is a popular catalyst of the MBH reaction. Virtually all the molecules contain an aziridine N as the catalytically active site, which is discovered by the GA since it is either not found in the initial population or discarded early only to be redisovered as the search progresses. Many of the GA searches also introduce a substituent with a hydrogen bond donor that helps to stabilize the transition state and thus lower the barrier. Two molecules are selected for further study based on their synthetic accessibility as predicted by the retrosynthesis package Manifold. For these two molecules we compute the entire free energy reaction profile at the DFT level and show that their rate determining barriers are 1.7 and 2.4 kcal/mol lower than that of DABCO. The molecule with the lowest barrier has higher barriers for the other steps compared to DABCO, but none of the barriers are competitive with the rate-determining barrier and is predicted to outperform DABCO. This demonstrates the power of free exploration of chemical a space compared to more constrained fragment-based approaches.

Nature Catalysis, Published online: 15 August 2022; doi:10.1038/s41929-022-00822-2

His-C2-directed modification is chemically challenging and rarely occurs in nature due to the low reactivity of this position. Now, the prenlytransferase LimF has been discovered and applied for geranylation of histidine-containing peptides and imidazole-containing small molecules, showcasing the versatility of this biocatalyst.

Benzaldehyde-derived iminium ions 1 form benzoxacyclic products 2 in a photochemical reaction cascade (n=0,1). Structurally different products 3 (meta photocycloaddition) and 4 (aza Paternò–Büchi reaction) are observed depending on two key substituents (R, R¹).

Abstract

While 2-alk-ω-enyloxy-sustituted benzaldehydes do not display any photochemical reactivity at the arene core, the respective iminium perchlorates were found to undergo efficient reactions either upon direct irradiation (λ=366 nm) or under sensitizing conditions (λ=420 nm, 2.5 mol% thioxanthen-9-one). Three pathways were found: (a) Most commonly, the reaction led to benzoxacyclic products in which the olefin in the tether underwent a formal, yet unprecedented carboformylation (13 examples, 44–99 % yield). The cascade process occurred with high diastereoselectivity and was found to be stereoconvergent. (b) If a substituent resides in the 3-position of the benzene ring, a meta photocycloaddition was observed which produced tetracyclic skeletons with five stereogenic centers in excellent regio- and diastereoselectivity (2 examples, 58–79 % yield). (c) If the tether was internally substituted at the alkene, an arene photocycloaddition was avoided and an azetidine was formed in an aza Paternò–Büchi reaction (2 examples, 95–98 % yield).

Water is utilized as benign electron-donor in photosynthesis, e.g. by cyanobacteria. Herein, a novel approach to shuttle them through the cyanobacterium's cell-membrane and to utilize them for extracellular NAD(P)H-dependent redox reactions is presented. The concept was demonstrated for 19 different reductive and oxidative enzymes from 4 different classes.

Abstract

Many biocatalytic redox reactions depend on the cofactor NAD(P)H, which may be provided by dedicated recycling systems. Exploiting light and water for NADPH-regeneration as it is performed, e.g. by cyanobacteria, is conceptually very appealing due to its high atom economy. However, the current use of cyanobacteria is limited, e.g. by challenging and time-consuming heterologous enzyme expression in cyanobacteria as well as limitations of substrate or product transport through the cell wall. Here we establish a transmembrane electron shuttling system propelled by the cyanobacterial photosynthesis to drive extracellular NAD(P)H-dependent redox reactions. The modular photo-electron shuttling (MPS) overcomes the need for cloning and problems associated with enzyme- or substrate-toxicity and substrate uptake. The MPS was demonstrated on four classes of enzymes with 19 enzymes and various types of substrates, reaching conversions of up to 99 % and giving products with >99 % optical purity.

Nanozymes are defined as nanomaterials with enzyme-like characteristics. Although tremendous effort has been devoted to developing nanozymes with high catalytic activity, the rational construction of nanozymes with high enantioselectivity remains a significant challenge. This Minireview provides an overview of recent developments in chiral nanozyme-based enantioselective biological catalysis, with an outlook on the opportunities and trends in this research area.

Abstract

As a neat combination of the characteristics of enzymatic activity and nanomaterials, nanozymes have attracted much attention. Although tremendous effort has been devoted to developing nanozymes with high catalytic activity, substrate selectivity is often overlooked in the construction of nanozymes. With their subtly evolving structures, natural enzymes generally possess high selectivity for chiral substrates which play important roles in biosynthesis and biomedical applications. However, the rational construction of nanozymes with high enantioselectivity remains a significant challenge. In this Minireview, we provide an overview of recent advances in strategies for the synthesis of chiral nanozymes and their enantioselective biological catalysis. We further focus on current challenges, potential solutions, and future developing trends of chiral nanozyme-based enantioselective biological catalysis. There is plenty of room to explore. This Minireview will provide new insights into the development of chiral nanozymes and their applications.

High-throughput μL-scale screenings revealed optimal conditions for in situ H₂O₂-generation using oxalate oxidase in bioconversions catalysed by unspecific peroxygenase. This enzymatic tandem exhibits extraordinary potential for selective C−H oxyfunctionalisation reactions of complex drug scaffolds.

Abstract

H₂O₂-driven enzymes are of great interest for industrial biotransformations. Herein, we show for the first time that oxalate oxidase (OXO) is an efficient in situ source of H₂O₂ for one of these biocatalysts, which is known as unspecific peroxygenase (UPO). OXO is reasonably robust, produces only CO₂ as a by-product and uses oxalate as a cheap sacrificial electron donor. UPO has significant potential as an industrial catalyst for selective C−H oxyfunctionalisations, as we confirm herein by testing a diverse drug panel using miniaturised high-throughput assays and mass spectrometry. 33 out of 64 drugs were converted in 5 μL-scale reactions by the UPO with OXO (conversion >70 % for 11 drugs). Furthermore, oxidation of the drug tolmetin was achieved on a 50 mg scale (TON_UPO 25 664) with 84 % yield, which was further improved via enzyme immobilization. This one-pot approach ensures adequate H₂O₂ levels, enabling rapid access to industrially relevant molecules that are difficult to obtain by other routes.

Nature Chemistry, Published online: 29 July 2022; doi:10.1038/s41557-022-01005-z

Burkholderia pseudomallei is a species of bacteria that poses a global health threat and, more generally, bacteria of the Burkholderia pseudomallei group cause severe diseases that are recalcitrant to treatment with antibiotics. Now, it has been shown how these infamous pathogens repurpose the widespread enzyme BurG to produce a reactive cyclopropanol head group found in the virulence-promoting malleicyprol toxins. Interrupting the synthesis of the cyclopropanol warhead is a potential route for developing antivirulence treatments.

Nature Catalysis, Published online: 22 July 2022; doi:10.1038/s41929-022-00813-3

General catalytic methods for free radical-mediated asymmetric transformations have long eluded synthetic organic chemists. Now, NAD(P)H-dependent ketoreductases are repurposed and engineered as highly efficient photoenzymes to catalyse asymmetric radical C–C couplings.

Artificial enzymes: Friedel-Crafts reactions are a mainstay methodology for organic chemists to construct aryl-alkyl and aryl-acyl linkages. Nature uses biocatalysts for performing such reactions to construct an array of bio-active compounds. In this Review we discuss several examples of such enzymes, as well Friedel-Crafts artificial enzymes and DNA catalysts, from the useful products they produce, to their mechanism and approaches for engineering.

Abstract

Friedel-Crafts alkylation and acylation reactions are important methodologies in synthetic and industrial chemistry for the construction of aryl-alkyl and aryl-acyl linkages that are ubiquitous in bioactive molecules. Nature also exploits these reactions in many biosynthetic processes. Much work has been done to expand the synthetic application of these enzymes to unnatural substrates through directed evolution. The promise of such biocatalysts is their potential to supersede inefficient and toxic chemical approaches to these reactions, with mild operating conditions - the hallmark of enzymes. Complementary work has created many bio-hybrid Friedel-Crafts catalysts consisting of chemical catalysts anchored into biomolecular scaffolds, which display many of the same desirable characteristics. In this Review, we summarise these efforts, focussing on both mechanistic aspects and synthetic considerations, concluding with an overview of the frontiers of this field and routes towards more efficient and benign Friedel-Crafts reactions for the future of humankind.

A new enantioselective version of the Nazarov cyclization is reported. The reaction design uses readily available alkenynones to trigger a gold(I)-catalyzed anti-Michael hydroarylation of the ynone followed by Nazarov cyclization. A chiral gold complex is able to control the absolute stereochemistry of the process. Cyclopenta[c]chromenones, which combine the 2H-chromene and cylopentanone cores, are synthesized with high yields and ee values.

Abstract

The asymmetric synthesis of cyclopentachromenones from gold-catalyzed reaction of readily available skipped alkenynones is described. This cascade reaction involves an initial anti-Michael hydroarylation of the ynone moiety to form a gold-functionalized dialkenylketone intermediate, followed by a Nazarov cyclization that proceeds in an unprecedented enantioselective manner. Excellent enantiomeric ratios and chemical yields are obtained under mild reaction conditions.

Nature Chemistry, Published online: 18 July 2022; doi:10.1038/s41557-022-00992-3

A genetically encoded phototrigger based on a xanthone amino acid can expand the scope of time-resolved serial femtosecond crystallography beyond naturally photoactive proteins. This approach has been used to uncover metastable reaction intermediates that occur prior to C–H bond activation in a human liver fatty-acid-binding protein mutant.