Biocatalysis@TUDelft

C-H functionalization provides unparalleled benefits for the late-stage modification of complex molecules. In recent years, photocatalysis has progressed significantly due to its mild conditions, sustainability and selectivities. Current mechanisms for photocatalytic C-H functionalization primarily involve direct single-electron oxidation and direct hydrogen atom transfer (d-HAT) by radicals, both of which are invalid for the functionalization of highly electron-deficient C(sp3)-H bonds. Here, we developed a cooperative photoenzymatic system consisting of a flavin-dependent ene-reductase (ER) and an exogenous photocatalyst fluorescein (FI) to achieve enantiodivergent functionalization of electron-deficient C(sp3)-H bonds. Mechanistic studies revealed a novel pathway for radical intermediate formation via excited-state FI*-induced single-electron oxidation of carbanions under alkaline conditions. The overall catalytic efficiency is enhanced by the electron transfer (ET) between FMNox and FI-*, while the stereoselectivity is controlled by ERs through enantioselective hydrogen atom transfer (HAT). This suggests that thermodynamically unfavorable single-electron oxidation of highly electron-deficient species can occur through ion-to-radical conversion. We anticipate that our novel mode of photoenzymatic catalysis will inspire new pathways for generating free radical intermediates and foster innovative strategies for achieving photoenzymatic new-to-nature reactions. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=75 SRC="FIGDIR/small/627421v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@d74f1eorg.highwire.dtl.DTLVardef@18b5cdaorg.highwire.dtl.DTLVardef@7d0fe1org.highwire.dtl.DTLVardef@9d9dda_HPS_FORMAT_FIGEXP M_FIG C_FIG

Bipyridyl-l-alanine (BpyAla) is a highly sought, metal-chelating non-canonical amino acid. However, its high cost has hindered many applications. Here, we develop a chemoenzymatic approach to efficiently construct BpyAla. This strategy is general to many types of metal-chelating amino acid, and we show that a newly available BpyAla analog can be incorporated into proteins using existing amber suppression technology.

Abstract

Metal-chelating noncanonical amino acids (ncAAs) are uniquely functional building blocks for proteins, peptide catalysts, and small molecule sensors. However, catalytic asymmetric approaches to synthesizing these molecules are hindered by their functional group variability and intrinsic propensity to ligate metals. In particular, bipyridyl-l-alanine (BpyAla) is a highly sought ncAA, but its complex, inefficient syntheses have limited utility. Here, we develop a chemoenzymatic approach to efficiently construct BpyAla. Three enzymes that can be produced in high titer together react to convert Gly and an aldehyde into the corresponding β-hydroxy ncAA, which is subsequently deoxygenated. We explore approaches to synthesizing biaryl aldehydes and show how the three-enzymatic cascade can access a range of α-amino acids with bulky side chains, including a variety of metal-chelating amino acids. We show that newly accessible BpyAla analogues are compatible with existing amber suppression technology, which will enable future merging of traditional synthetic and biosynthetic approaches to tuning metal reactivity.

The unspecific peroxygenase rAaeUPO-PaDa-I-H catalyses the oxidation of Z- and E-allylic alcohols with complementary selectivity, giving epoxide and aldehyde/acid products, respectively. Both reactions were performed on a preparative scale with yields of up to 80 %, and the epoxidations proceed with excellent enantioselectivity (99 % ee).

Abstract

Unspecific peroxygenases (UPOs) catalyze the selective oxygenation of organic substrates using only hydrogen peroxide as the external oxidant. The PaDa−I variant of the UPO from Agrocybe aegerita catalyses the oxidation of Z- and E-allylic alcohols with complementary selectivity, giving epoxide and carboxylic acid/aldehyde products respectively. Both reactions can be performed on preparative scale with isolated yields up to 80 %, and the epoxidations proceed with excellent enantioselectivity (>99 % ee). The divergent reactions can also be used to transform E/Z mixtures of allylic alcohols, enabling both product series to be isolated from a single reaction. The utility of the epoxidation method is exemplified in the total synthesis of both enantiomers of the insect pheromone disparlure, including a highly enantioselective gram-scale transformation. These reactions provide further evidence for the potential of UPOs as catalysts for the scalable preparation of important oxygenated intermediates.

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.

Science, Volume 386, Issue 6722, November 2024.

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.

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.

Science, Volume 386, Issue 6723, November 2024.

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.

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

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

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

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

Synergistic photobiocatalysis for enantioselective triple radical sorting

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.

Biocatalysts are prized for their selectivity, tunability, and their compatibility with environmentally-friendly reaction conditions. Introduction of unnatural cofactors opens the door to new reactive enzymatic intermediates, and in turn, the possibility for new biochemical reactions. In the present study, we employed a de novo biosynthesized, non-natural cofactor, cobalt protoporphyrin IX,1 to generate a mono-nuclear cobalt hydride intermediate in the active site of a common P450 scaffold. We show that this cobalt hydride intermediate engages in metal-hydrogen atom transfer (M-HAT) reactivity, a well-studied and highly utilized reactivity pattern in synthetic chemistry,2 but which is not known to operate in nature. Because the required cofactor is fully biosynthesized and incorporated into proteins in vivo, the catalysts are highly amendable to directed evolution. We leveraged the ability to quickly access these new artificial metalloenzymes with a colorimetric screen and evolved new variants for M-HAT-mediated deallylation of phenols. We showed how common silanes have a propensity to hydrolysis that can be overcome with directed evolution by accelerating metal-hydride formation from a bulky, water stable silane. During this evolution, we discovered that variants were catalyzing HAT to the colorimetric probe itself, resulting in a unique reductive dearomatization reaction. This radical process occurs efficiently under aerobic conditions and reactions of this type have not been observed previously. These discoveries demonstrate how the tunability of biocatalytic systems can enable innovations in synthetic chemistry. We anticipate that further engineering and study of M-HAT biocatalysts will prompt new questions about hydrogen atom transfer reactivity and enable the adoption of biocatalysts for numerous synthetically useful transformations.

The high-yielding fungal peroxygenase, MroUPO-TN, catalyzes the regioselective remote-site functionalization of hydrocarbons, providing useful and new synthetic building blocks in an economical and sustainable process. Bromocyclooctane affords 4-bromocyclooctanone in 80% yield, whereas 1-haloalkanes are transformed into the corresponding ω-1 haloketones. Deuterium labeling and ¹⁸O-labeling experiments show that the selectivity for 4-halocyclooctanones derives from selective, remote site oxygenation.

Abstract

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.

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.

The vanadium-dependent haloperoxidase (VHPO) class of enzymes are discovered as an effective biocatalyst platform for nitrogen-halogen (N−X) bond formation. VHPOs perform selective halogenation on a range of substituted benzamidine hydrochlorides to produce the corresponding N’-halobenzimidamides and this technology is applied to 1,2,4-oxadiazole synthesis.

Abstract

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.

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.

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.