R.B. Leveson-Gower

We report the development of an engineered P450 monooxygenase that mediates a chemo- and stereo-selective alkene epoxidation to generate a key chiral precursor of the anti-tuberculosis drug delamanid. Screening of an in-house P450 monooxygenase panel led to the identification of a BM3 variant, containing five mutations, with activity for the target transformation. Over a single round of laboratory evolution and gene shuffling, three further beneficial mutations were introduced leading to an order of magnitude increase in the de-sired activity, with a total turnover number (TON) of >3000. This newly engineered enzyme generates a chiral epoxide intermediate from an alkene precursor in a single step with 98% e.e. and >97% conversion. Initial efforts to scale the biocatalytic transformation high-lights the potential of the engineered enzyme to provide a more efficient and sustainable route for the manufacture of delamanid.

Nature, Published online: 21 November 2024; doi:10.1038/s41586-024-08399-5

Synergistic photobiocatalysis for enantioselective triple radical sorting

Nature, Published online: 20 November 2024; doi:10.1038/s41586-024-08327-7

Photocatalytic C–F bond activation in small molecules and polyfluoroalkyl substances

New enzymes can be designed by starting from a description of an ideal active site composed of catalytic residues surrounding the reaction transition state(s) and identifying or generating a protein scaffold that supports the site1-7, but there are a few current limitations. First, the catalytic efficiencies achieved by such efforts have generally been quite low, and considerable optimization by directed evolution has been required to reach activities typical of native enzymes.8-10 Second, generative AI methods such as RFdiffusion11,12 now enable the direct generation of proteins around active sites, but to date, such scaffolding has required specification of both the position in the sequence and the backbone coordinates of the catalytic residue, which complicates sampling. Here we introduce a generative AI method called RFam that overcomes these limitations and use it to design zinc metallohydrolases starting from a density functional theory description of active site geometry. Of 96 designs tested experimentally, the most active has a kcat/KM of 23,000 M-1 s-1, orders of magnitude higher than previously designed metallohydrolases.6,7,13,14 This 148 amino acid protein has a novel fold with an enclosed chamber which positions the reaction substrate nearly perfectly for attack by a catalytic water molecule activated by the bound metal and is predicted by ChemNet15 to have a highly preorganized active site. The ability to generate high activity catalysts starting from quantum chemistry calculated active site geometries without experimental optimization should open the door to a new generation of potent designer enzymes.16,17

Functionalisation of nucleosides at the 2'-position has become an important modification for therapeutic purposes to tailor pharmacological properties. Chemical synthesis of these molecules is challenging, and recent studies have explored bottom-up strategies with enzymes of the nucleoside salvage pathway and late-stage functionalisation capabilities. More than 55 years ago, a pyrimidine nucleoside 2'-hydroxylase (PDN2'H) activity was described in three fungal species. However, the corresponding protein sequences have never been reported. This study describes the identification and characterisation of PDN2'H from Neurospora crassa, which naturally hydroxylates thymidine at the 2'-position as now verified by NMR. Site-directed mutagenesis confirmed the protein to be an α-ketoglutarate-/Fe(II)-dependent dioxygenase. We performed investigation of its substrate scope, phylogeny, thermostability and elucidated the enzymatic mechanism with help of PDN2'H’s crystal structure co-crystallised with thymidine. This work adds a long sought-after and important nucleoside-modifying protein to the biocatalytic portfolio.

Science, Volume 386, Issue 6723, November 2024.

The design of proteins with tailored functions is of immense interest to biotechnology, medicine, and the chemical industry. While protein design is rapidly evolving with the use of AI techniques, the design of complex enzymes remains a challenge. Here, we present the use of two large language models (LLMs), ZymCTRL and ProtGPT2, for the generation of de novo enzymes that catalyze the triosephosphate isomerase (TIM) reaction. Natural TIM enzymes are obligatory oligomers that catalyze a multi-step isomerization reaction near the diffusion limit. This makes TIM an ideal target to assess the generative ability of protein language models. Newly generated sequences were filtered to obtain a set of twelve candidates from each approach for experimental validation. Multiple constructs from both language models exhibit the intended function in vivo through their ability to complement a TIM-deficient E. coli strain. In-depth characterization of the best-behaving artificial enzyme reveals behavior and catalytic efficiency close to its natural counterparts. These findings support the use of conditional and fine-tuned unconditional LLMs for the generation of complex enzymes.

Transition metal–hydrides have been widely exploited in homogenous catalysis for hydrofunctionalization of unsaturated moieties, including carbonyls, alkenes and alkynes. As a complement to the well-established chemistry of these complexes involving heterolytic metal–hydride bond cleavage, metal–hydride hydrogen atom transfer (MHAT) has attracted increased interest, as it offers a promising strategy for radical hydrofunctionalziation of unactivated alkenes thus enabling late-stage diversification of complex molecules. However, due the weak interactions between the prochiral organic radical species and the enantiopure metal catalyst, achieving asymmetric MHAT remains challenging. Herein, we report our efforts to repurpose cytochrome P450 enzymes to catalyze asymmetric MHAT, a new-to-nature reaction. Directed evolution of the well-studied P450BM3 (CYP102A1) enzyme led to the identification of a triple mutant that catalyzes asymmetric MHAT radical cyclization of unactivated alkenes to afford diverse cyclic compounds, including pyrrolidines, in up to a 97:3 enantiomeric ratio under aerobic whole cell conditions. Mechanistic investigations support an MHAT mechanism proceeding via homolytic cleavage of a fleeting iron(III)hydride species. Directed evolution using CYP119 as hemoprotein scaffold led to the identification of a stereocomplementary MHATase, highlighting the potential of repurposed hemoproteins for MHAT biocatalysis. Our study showcases the potential of integrating abiotic metal–hydride activity into native metalloenzymes to expand the scope of asymmetric radical biocatalysis.

Science, Volume 386, Issue 6722, November 2024.

Nature Synthesis, Published online: 05 November 2024; doi:10.1038/s44160-024-00671-w

The chemical synthesis of nucleoside analogues with modifications at the 2-position often requires multiple steps and the extensive use of protecting groups. Now, biocatalytic cascades are reported for the synthesis of 2-functionalized sugars and 2′-functionalized nucleosides, using enzymes derived from those of the purine nucleoside salvage pathway.

Nature Communications, Published online: 28 November 2024; doi:10.1038/s41467-024-54613-3

There is a paucity of studies on enzyme stereoselectivity from an evolutionary biochemistry perspective. Here, the authors use ancestral sequence reconstruction to trace the evolution of stereoselectivity in imine reductases, elucidate its structural basis, and investigating the role of epistasis.

Nature Chemistry, Published online: 26 November 2024; doi:10.1038/s41557-024-01671-1

Expanding the biocatalysis toolbox for selective desaturation is of great value. Now ‘ene’-reductases have been repurposed to mediate dehydrogenation, the reverse process of their native activity. The developed biocatalytic desaturation platform enables desymmetrizing desaturation of cyclohexanones for the synthesis of diverse cyclohexenones that bear a remote quaternary stereogenic centre.

The generic incorporation of a thioxanthone-containing amino acid into a protein scaffold is described. The resulting artificial photoenzyme was engineered to catalyze a dearomative [2+2] cycloaddition reaction in high yields with excellent enantioselectivity.

Abstract

Genetically encodable photosensitizers allow the design of artificial photoenzymes to expand the scope of abiological reactions. Herein, we report the genetic incorporation of a thioxanthone-containing amino acid into a protein scaffold via an engineered pyrrolysyl-tRNA/pyrrolysyl-tRNA synthetase pair. The designer enzyme was engineered to catalyze a dearomative [2+2] cycloaddition reaction in high yields (up to>99 % yield) with excellent enantioselectivity (up to 98 : 2 e.r.). This work provides a robust and facile method for photoenzyme design and lays the foundation for the development of further photoenzymatic reactions.

Mn(salen)-based artificial metalloenzymes (ArMs) were constructed by embedding biotinylated Mn(salen) complexes into streptavidin. Their activities were improved by genetic optimization of protein scaffold and the ArM variants catalyzed the aziridination of styrene and oxidation of benzylic C−H bonds with up to 19 and 146 turnover numbers.

Abstract

The development of artificial metalloenzymes (ArMs) offers a potent approach to incorporate non-natural chemical reactions into biocatalysis. Here we report the assembly of Mn(salen)-based ArMs by embedding biotinylated Mn(salen) complexes into streptavidin (Sav) variants. Using commercially available nitrene and oxo transfer reagents, these biohybrid catalysts catalyzed the aziridination of alkenes and oxidation of benzylic C−H bonds with up to 19 and 146 turnover numbers.

Science, Volume 386, Issue 6728, Page 1421-1427, December 2024.

Nature Catalysis, Published online: 20 December 2024; doi:10.1038/s41929-024-01263-9

Photoredox catalysis is merged with metalloenzymatic catalysis to enable asymmetric decarboxylative azidation and thiocyanation. These transformations are achieved by coupling the photoredox activation of N-hydroxyphthalimide esters using a synthetic photocatalyst with enantioselective radical capture by Fe(iii) intermediates of non-haem iron enzymes.

Lipopolysaccharides (LPS) play important roles in the Gram-negative bacterial cell envelope. LPS are located in the outer leaflet of the outer membrane and generally serve as the first defense layer against environmental stress. 3-Deoxy-D-manno-oct-2-ulosonic acid (Kdo) is a highly conserved monosaccharide that resides in the inner core region of LPS and that connects the lipid A moiety to the extending polysaccharide chain through the hydroxyl group on its C-5 position. Due to its central function in LPS, we hypothesized that metabolically incorporated Kdo derivatives modified on the C-5 position may impair LPS synthesis and therefore lead to a reduced outer membrane integrity. To test this, we successfully synthesized four Kdo derivatives, 5-epi-Kdo, 5-deoxy-Kdo, 5-epi-Kdo-8-N3, and 5-deoxy-Kdo-8-N3, and incubated Escherichia coli (E. coli) strains in the presence of these derivatives to investigate their influence on LPS production and labeling. Interestingly, while the 5-deoxy derivatives were not incorporated, 5-epi-Kdo-8-N3 was suc-cessfully incorporated in cell envelope-associated LPS and increased the sensitivity of the bacteria to vancomycin, indi-cating that 5-epi-Kdo-8-N3 incorporation in LPS interferes with outer membrane integrity.

Fullerenes, spherical molecules made entirely of carbon atoms, have played a foundational role in the birth of nanoscience. Despite extensive research, however, comparable structures composed of other elements remain elusive, highlighting the unique bonding properties of carbon that enable the formation of such remarkable nanoscale architectures. Here, we report the discovery of a fullerene-like 80-atom boron cluster using photoelectron spectroscopy and quantum-chemical calculations. The photoelectron spectrum of B80– reveals a surprisingly simple spectral pattern, suggesting a high symmetry B80 cluster with a large energy gap. Following extensive structural searches and high-level ab initio calculations, we find that a spherical B80– represents the global minimum with only its simulated spectrum agreeing with the experimental result. We show that the electronic structure and chemical bonding of B80 closely mimic those of the C60 buckminsterfullerene. The discovery of the B80 buckminsterfullerene will stimulate its bulk synthesis and pave the way for the development of boron-fullerene chemistry.

Nature Communications, Published online: 18 December 2024; doi:10.1038/s41467-024-55212-y

Affinity chromatography allows for the separation of biomolecules such as proteins, based on a change in the chemical solvent composition and the resulting impacts on ligand binding. Here, authors introduce a physical principle by exploiting the light-dependent interaction between the Azo-tag and an α- CD chromatography matrix.

Enzymes are attractive catalysts due to their high chemo-, regio-, and enantio-selectivity. In recent years the application of enzymes in organic synthesis has expanded dramatically, especially for the synthesis of chiral alcohols and amines, two very important functional groups found in many active pharmaceutical ingredients (APIs). Indeed, many elegant routes employing such compounds have been described by industry. Yet, for the synthesis chiral thiols and thioethers, likewise found in APIs albeit less ubiquitous, only very few biocatalytic syntheses have been reported, and stereo-control has proved challenging. Here we apply ene-reductases (EREDs), whose ability to initiate and control chemically challenging radical chemistries has recently emerged, to the synthesis of chiral thioethers from α-bromoacetophenones and pro-chiral vinyl sulfides, without requiring light. Depending on the choice of ERED either enantiomer of the product could be accessed. Highest conversion and selectivity were achieved with GluER T36A using fluorinated substrates, reaching up to 82% conversion and >99.5% ee. With α-bromoacetophenone and α-(methylthio)styrene, the reaction could be performed on a 100 mg scale, affording the product in 46% isolated yield with 93% ee. Finally, mechanistic studies were carried out using stopped-flow spectroscopy and protein mass-spectrometry, providing insight into the preference of the enzyme for the inter-molecular reaction. This work paves the way for new routes for the synthesis of thioether-containing compounds.

Oxazolidinones are important heterocycles widely utilized in medicinal chemistry for the synthesis of antifungals, antibacterials, and other bioactive compounds and in organic chemistry as chiral auxiliaries for asymmetric synthesis. Herein, we report a biocatalytic strategy for the synthesis of enantioenriched oxazolidinones through the intramolecular C(sp3)‑H amination of carbamate derivatives using engineered myoglobin-based catalysts. This method is applicable to a diverse range of substrates, with high functional group tolerance, providing enantioenriched oxazolidinones in good yields and with high enantioselectivity. The synthetic utility of this methodology is further highlighted by the development of enantiodivergent biocatalysts for this transformation and through the preparative-scale synthesis of key oxazolidinone intermediates for the production of the cholesterol-lowering drugs Ezetimibe and CJ-15-161. An outer sphere mutation, Y146F, was found to be beneficial to favor the productive C–H amination reaction over an unproductive reductive pathway commonly observed in hemeprotein-catalyzed nitrene transfer reactions. This study demonstrates a biocatalytic, enantiodivergent synthesis of oxazolidinones via C-H amination of carbamate derivatives, offering an attractive strategy for the synthesis of these valuable intermediates for applications in medicinal chemistry, target-directed synthesis, and asymmetric synthesis.