R.B. Leveson-Gower

Cost-effective electrocatalysts is a key constituent to establish the balance of cost and catalytic efficiency for oxygen evolution reaction (OER) via water electrolysis in the area of energy conversion and storage. NiFe phosphide decorated with trace amount of iridium (Ir) species in-situ grown on carbon cloth was prepared by a facile wet chemistry approach followed by a phosphorization post-treatment at a relative low temperature. The optimal electrocatalyst, Ir2-NiFePx/CC, exhibits excellent OER activity, with an low overpotential of 190 mV at 10 mA cm–2 for alkaline OER, and a desirable long-term durability over 90 h. The outstanding OER performance stems from the structural evolution via phosphorization process, Ir decoration with more high-valence stated Ir4+ species, and tight connection between individual components of the electrode, which gives rise to the strong activity to the active sites and faster reaction kinetics in the alkaline OER process. Mover, the Ir loading was as low as approximately ~1.7 wt% (0.29 mg cm-2), showing promissing propective in cost-effective OER.

We describe the discovery of an unspecific peroxygenase (UPO) variant that catalyzes the remote-site functionalization of halogenated and unsaturated hydrocarbons with high catalytic site-specificity. UPOs are fungal heme-thiolate biocatalysts with wide-ranging oxidative activities, including C–H bond oxygenation, usually with limited regioselectivity. We describe here a wild-type MroUPO, newly isolated in high yield from a previously uncharacterized strain of Marasmius rotula. This variant, MroUPO-TN, catalyzes the selective oxygenation of a range of haloalkanes, cyclic haloalkanes and cyclic olefins to generate useful remote-site haloketones. The regioselectivity for eight-membered rings reaches 99% with significant enantiomeric excess. Mechanistic studies performed with deuterated substrates and 18O-labeling experiments have revealed a synergy between intrinsic substrate properties and the highly aliphatic, heme active site. The observed selectivity offers routes to new and useful, bifunctional synthons and pharmacophores, thus providing practical ways to employ these natural and environmentally benign biocatalysts.

Science Advances, Volume 10, Issue 40, October 2024.

The evolution of a promiscuous enzyme for its various activities often results in catalytically specialized variants. This is an important natural mechanism to ensure the proper functioning of natural metabolic networks. It also acts as both a curse and blessing for enzyme engineers, where enzymes that have undergone directed evolution may exhibit exquisite selectivity at the expense of a diminished overall catalytic repertoire. We previously performed two independent directed evolution campaigns on a promiscuous artificial enzyme that leverages the unique properties of a non-canonical amino acid (ncAA) para- aminophenylalanine (pAF) as catalytic residue, resulting in two evolved variants which are both catalytically specialized. Here, we combine mutagenesis, crystallography and computation to reveal the molecular basis of the specialization phenomenon. In one evolved variant, an unexpected change in quaternary structure biases substrate dynamics to promote enantioselective catalysis, whilst the other demonstrates synergistic cooperation between natural side chains and the pAF residue to form semi-synthetic catalytic machinery. Our analysis provides valuable insights for the future engineering of effective artificial enzymes which employ either the widely used LmrR scaffold or pAF catalytic residue.

Enzymes that proceed through multistep reaction mechanisms often utilize complex, polar active sites positioned with sub-angstrom precision to mediate distinct chemical steps, which makes their de novo construction extremely challenging. We sought to overcome this challenge using the classic catalytic triad and oxyanion hole of serine hydrolases as a model system. We used RFdiffusion1 to generate proteins housing catalytic sites of increasing complexity and varying geometry, and a newly developed ensemble generation method called ChemNet to assess active site geometry and preorganization at each step of the reaction. Experimental characterization revealed novel serine hydrolases that catalyze ester hydrolysis with catalytic efficiencies (kcat/Km) up to 3.8 x 103 M-1 s-1, closely match the design models (C RMSDs < 1 [A]), and have folds distinct from natural serine hydrolases. In silico selection of designs based on active site preorganization across the reaction coordinate considerably increased success rates, enabling identification of new catalysts in screens of as few as 20 designs. Our de novo buildup approach provides insight into the geometric determinants of catalysis that complements what can be obtained from structural and mutational studies of native enzymes (in which catalytic group geometry and active site makeup cannot be so systematically varied), and provides a roadmap for the design of industrially relevant serine hydrolases and, more generally, for designing complex enzymes that catalyze multi-step transformations.

Nature Chemistry, Published online: 28 August 2024; doi:10.1038/s41557-024-01608-8

Catalytic asymmetric radical dearomatization has remained a daunting task due to the challenges in exerting stereocontrol over highly reactive radical intermediates. Now, using metalloredox biocatalysis, new-to-nature radical dearomatases P450rad1–P450rad5 have been engineered to facilitate asymmetric dearomatization of a broad spectrum of aromatic substrates, including indoles, pyrroles and phenols.

Nitrogen-containing compounds are valuable synthetic intermediates and targets in nearly every chemical industry. While methods for nitrogen-carbon and nitrogen-heteroatom bond formation have primarily relied on nucleophilic nitrogen atom reactivity, molecules containing nitrogen-halogen bonds allow for electrophilic or radical reactivity modes at the nitrogen center. Despite the growing synthetic utility of nitrogen-halogen bond-containing compounds, selective catalytic strategies for their synthesis are largely underexplored. We recently discovered that the vanadium-dependent haloperoxidase (VHPO) class of enzymes are a suitable biocatalyst platform for nitrogen-halogen bond formation. Herein, we show that VHPOs perform selective halogenation of a range of substituted benzamidine hydrochlorides to produce the corresponding N’-halobenzimidamides. This biocatalytic platform is applied to the synthesis of 1,2,4-oxadiazoles from the corresponding N-acylbenzamidines in high yield and with excellent chemoselectivity. Finally, the synthetic applicability of this biotechnology is demonstrated in an extension to nitrogen-nitrogen bond formation and the chemoenzymatic synthesis of the Duchenne muscular dystrophy drug, ataluren.

The catalytic repertoire of nature has been expanded over the past decades by the introduction of artificial metalloenzymes. These are enzymes containing a synthetic metal complex or a non-native metal ion. However, combining noble metal catalysis and enzymes remains challenging due to the lack of suitable ligands to bind these complexes. So far, noble metal artificial metalloenzyme design mostly involves in vitro approaches of ligand anchoring, like covalent modification of a cysteine residue or via supramolecular assembly. Here, we show a facile strategy to anchor a variety of 4d and 5d-transition metal complexes via genetic incorporation of a thiophenolic metal-binding ligand. We created a methodology to efficiently incorporate 4-mercaptophenylalanine in a protein scaffold using the stop codon suppression technology. The incorporated non-canonical amino acid was capable of binding a variety of noble metal complexes. To showcase the catalytic applications of this methodology, we developed an artificial hydroaminase by binding gold ions to the thiophenol-containing protein. The benefit of in vivo incorporation of the ligand is demonstrated by the susceptibility of catalytic activity to the microenvironment around the metal site, which can be modulated by changing the position of the ligand within the protein or by mutation of residues in its proximity.

Nature Chemistry, Published online: 19 July 2024; doi:10.1038/s41557-024-01562-5

Although natural terpenoid cyclases generate polycyclic structures through cationic intermediates, alternative radical cyclization pathways are underexplored. Now an artificial radical cyclase has been prepared by anchoring a biotinylated cobalt Schiff-base complex within a chimeric streptavidin scaffold. Chemogenetic optimization of the catalytic performance affords enantioenriched terpenoids via a metal-catalysed H-atom transfer mechanism.

Artificial enzymes that operate with pnictogen bonds or σ-hole interactions in general are introduced: Transfer hydrogenation of quinolines accelerates with biotinylated pnictogen-bonding cofactors and their interfacing with streptavidin and mutants, shows saturation behavior with transition-state recognition three orders of magnitude beyond substrate recognition, and the emergence of stereoselectivity.

Abstract

The objective of this study was to create artificial enzymes that capitalize on pnictogen bonding, a σ-hole interaction that is essentially absent in biocatalysis. For this purpose, stibine catalysts were equipped with a biotin derivative and combined with streptavidin mutants to identify an efficient transfer hydrogenation catalyst for the reduction of a fluorogenic quinoline substrate. Increased catalytic activity from wild-type streptavidin to the best mutants coincides with the depth of the σ hole on the Sb(V) center, and the emergence of saturation kinetic behavior. Michaelis–Menten analysis reveals transition-state recognition in the low micromolar range, more than three orders of magnitude stronger than the millimolar substrate recognition. Carboxylates preferred by the best mutants contribute to transition-state recognition by hydrogen-bonded ion pairing and anion-π interactions with the emerging pyridinium product. The emergence of challenging stereoselectivity in aqueous systems further emphasizes compatibility of pnictogen bonding with higher order systems catalysis.

“If I could be granted a superpower, it would be the ability to fluently speak and understand every language (especially German) in the world because it would allow me to connect with people from all cultures and backgrounds … My group has fun by challenging each other in basketball matches, fostering friendly competition and team spirit both in and out of the lab …” Find out more about Babis Pappas in his Introducing… Profile.

Nature Communications, Published online: 09 July 2024; doi:10.1038/s41467-024-50141-2

Exploring the promiscuity of native enzymes is a promising strategy for expanding their synthetic applications. Here, the authors show that old yellow enzymes (OYEs) can facilitate the Morita-Baylis-Hillman reaction (MBH reaction), leveraging substrate similarities between MBH reaction and reduction, and engineer GkOYE.8 with no reduction activity, but enhanced MBH activity.

Medicinal chemists in the modern era are targeting molecules with greater complexity to address increasingly challenging biological targets, a drive to enhance on-target specificity as well as physiochemical properties. As such, structures with greater fraction sp3 (Fsp3) character, reminiscent to those found in nature, are being synthesized. Many decades of synthetic methodology development have democratized access to flat, high sp2 (for example biaryl linkages) which has led to the commercialization of innumerable medicines. Those approaches rely heavily on electrophilic aromatic substitution (such as halogenation) followed by Pd-based cross coupling. In contrast, methods and strategies that allow for similarly modular and rapid construction of three-dimensional saturated molecules are less well developed. Here we exemplify a new approach for the rapid, modular, enantioselective construction of piperidine frameworks (the saturated analog of pyridine) that combines robust, tunable, and scalable biocatalytic methods with the logic of radical cross coupling. Thus, a set of reliable enzymatic systems (analogous to site-selective aromatic functionalization) provides scalable access to enantiopure hydroxyacid- containing piperidine derivatives that can be utilized to dramatically simplify routes to medicinally important molecules and natural products by employing recently developed electrocatalytic couplings (analogous to Pd-based cross couplings in aromatic systems). This study points to a different approach to rapidly access complex architectures that may appeal to both medicinal and process chemists alike.

Nature, Published online: 26 June 2024; doi:10.1038/d41586-024-02105-1

Prioritizing family life has earned me respect in my field — and my research has improved, too, says Dritjon Gruda.

Nature, Published online: 19 June 2024; doi:10.1038/s41586-024-07601-y

A deep learning approach enables accurate computational design of soluble and functional analogues of membrane proteins, expanding the soluble protein fold space and facilitating new approaches to drug screening and design.

The remarkable efficiency with which enzymes catalyze small molecule reactions has driven their widespread application in organic chemistry. Here, we employ automated fast-flow solid-phase synthesis to access full-length enzymes without restrictions on the number and structure of non-canonical amino acids incorporated. We demonstrate the total syntheses of Fe-dependent Bacillus subtilis myoglobin (BsMb) and sperm whale myoglobin (SwMb), which displayed excellent enantioselectivity and yield in carbene transfer reactions. Absolute control over enantioselectivity in styrene cyclopropanation was achieved using L- and D-BsMb mutants which delivered each enantiomer of cyclopropane product in identical and opposite enantiomeric enrichment. BsMb mutants outfitted with non-canonical amino acids were used to facilitate detailed structure-activity relationship studies, revealing a previously unrecognized hydrogen-bonding interaction as the primary driver of enantioselectivity in styrene cyclopropanation.

The discovery of small-molecule agents for chemical biology and therapeutic applications depends upon the ability to access and explore new biologically relevant regions of chemical space, a goal often pursued through diversity-oriented synthesis (DOS). In this report, we describe the design and implementation of P450-mediated chemoenzymatic diversity-oriented synthesis (CeDOS), a strategy that leverages chemo- and regiodivergent P450-catalyzed oxyfunctionalizations as key steps for enabling the synthesis of complex molecules that resemble natural products, a major source of bioactive molecules and drugs. Using this strategy, a library of over 50 novel and structurally diverse natural product-like compounds was generated through skeletal rearrangement and diversification of a plant-derived terpene via divergent chemoenzymatic routes enabled by selective C–H hydroxylation and epoxidation reactions catalyzed by engineered P450s. This CeDOS library encompass many unique and unprecedented organic scaffolds, many of which were determined to exhibit notable cytotoxicity against human cancer cells as well as diversified anticancer activity profiles. This work demonstrates the power of the present chemoenzymatic diversity-oriented synthesis strategy for directing the construction and discovery of novel bioactive molecules and it offers a blueprint for the broader application of this approach toward the creation and exploration of natural product-like chemical libraries.