Engineering Hydroxylase Activity, Selectivity, and Stability for a Scalable Concise Synthesis of a Key Intermediate to Belzutifan

Angewandte Chemie International Edition, Journal Year: 2024, Volume and Issue: 63(13)

Published: Jan. 27, 2024

Biocatalytic oxidations are an emerging technology for selective C-H bond activation. While promising a range of oxidations, practical use enzymes catalyzing aerobic hydroxylation is presently limited by their substrate scope and stability under industrially relevant conditions. Here, we report the engineering application non-heme iron α-ketoglutarate-dependent dioxygenase direct stereo- regio-selective non-native fluoroindanone en route to oncology treatment belzutifan, replacing five-step chemical synthesis with enantioselective hydroxylation. Mechanistic studies indicated that formation desired product was enzyme overoxidation, these properties subsequently improved directed evolution, yielding biocatalyst capable >15,000 total turnovers. Highlighting industrial utility this biocatalyst, high-yielding, green, efficient oxidation demonstrated at kilogram scale belzutifan.

Language: Английский

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Engineering non-haem iron enzymes for enantioselective C(sp3)–F bond formation via radical fluorine transfer

Nature Synthesis, Journal Year: 2024, Volume and Issue: 3(8), P. 958 - 966

Published: March 28, 2024

Language: Английский

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Regioselective Radical Alkylation of Arenes Using Evolved Photoenzymes

Journal of the American Chemical Society, Journal Year: 2023, Volume and Issue: 145(21), P. 11866 - 11874

Published: May 18, 2023

Substituted arenes are ubiquitous in molecules with medicinal functions, making their synthesis a critical consideration when designing synthetic routes. Regioselective C–H functionalization reactions attractive for preparing alkylated arenes; however, the selectivity of existing methods is modest and primarily governed by substrate's electronic properties. Here, we demonstrate biocatalyst-controlled method regioselective alkylation electron-rich electron-deficient heteroarenes. Starting from an unselective "ene"-reductase (ERED) (GluER-T36A), evolved variant that selectively alkylates C4 position indole, elusive using prior technologies. Mechanistic studies across evolutionary series indicate changes to protein active site alter character charge transfer (CT) complex responsible radical formation. This resulted significant degree ground-state CT complex. on C2-selective ERED suggest evolution GluER-T36A helps disfavor competing mechanistic pathway. Additional engineering campaigns were carried out C8-selective quinoline alkylation. study highlights opportunity use enzymes reactions, where small molecule catalysts struggle selectivity.

Language: Английский

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Discovery and substrate specificity engineering of nucleotide halogenases

Nature Communications, Journal Year: 2024, Volume and Issue: 15(1)

Published: June 19, 2024

Abstract C2′-halogenation has been recognized as an essential modification to enhance the drug-like properties of nucleotide analogs. The direct C2ʹ-halogenation 2′-deoxyadenosine-5′-monophosphate (dAMP) recently achieved using Fe(II)/α-ketoglutarate-dependent halogenase AdaV. However, limited substrate scope this enzyme hampers its broader applications. In study, we report two halogenases capable halogenating 2ʹ-deoxyguanosine monophosphate (dGMP), thereby expanding family halogenases. Computational studies reveal that specificity is regulated by binding pose phosphate group. Based on these findings, successfully engineered mutating second-sphere residues. This work expands toolbox and provides insights into regulation mechanism specificity.

Language: Английский

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New insights into exploring new functional enzymes through the enzyme promiscuity

International Journal of Biological Macromolecules, Journal Year: 2025, Volume and Issue: 304, P. 140576 - 140576

Published: Feb. 2, 2025

Language: Английский

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Protecting group-free introduction of amino acids to polymers through the aza-Michael reaction

Polymer Chemistry, Journal Year: 2024, Volume and Issue: 15(11), P. 1123 - 1132

Published: Jan. 1, 2024

The incorporation of natural amino acids onto a silicone framework is efficiently and simply achieved, without protecting groups or solvents, by using aza-Michael addition to acrylate-modified silicones.

Language: Английский

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Electrostatically Regulated Active Site Assembly Governs Reactivity in Nonheme Iron Halogenases

ACS Catalysis, Journal Year: 2023, Volume and Issue: 13(20), P. 13743 - 13755

Published: Oct. 11, 2023

Non-heme iron halogenases (NHFe-Hals) catalyze the direct insertion of a chloride ion at an unactivated carbon position using high-valent haloferryl intermediate. Despite more than decade structural and mechanistic characterization, rigorous understanding entire catalytic cycle NHFe-Hals how they facilitate binding, activation, reactivity with specific substrates functionalizing anions remains unclear. Here, we focus on binding active site assembly in freestanding halogenases, BesD HalB, which directly chlorination lysine without need for partner protein. While affinities to BesD's sites are extremely weak (Kd values ∼50 560 mM, respectively), demonstrate strong positive heterotropic cooperativity (cooperativity constant, α ∼ 15,500) between events such that bind efficiently when simultaneously present physiologically relevant concentrations. Using combination computational rational protein design studies, identify negatively charged residue, E119, locks unless both anion positively substrate present. Removing this electrostatic lock by mutating E119 polar/neutral glutamine alanine residues results 6.7- 14-fold increase affinity anion, respectively. A concomitant order magnitude decrease yields is observed as impaired these mutants, yet chemoselectivity profile rather similar. Beyond implications overall performance, show electrostatically regulated stage can also determine its promiscuity C–H functionalization other bromide, azide, nitrite. Overall, our studies highlight complex effects play during like along their performance BesD-like halogenases.

Language: Английский

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Controllable Multihalogenation of a Non-native Substrate by the SyrB2 Iron Halogenase

ACS Catalysis, Journal Year: 2024, Volume and Issue: 14(17), P. 13209 - 13218

Published: Aug. 19, 2024

Geminal, multihalogenated functional groups are widespread in natural products and pharmaceuticals, yet no synthetic methodologies exist that enable selective multihalogenation of unactivated C–H bonds. Biocatalysts powerful tools for late-stage functionalization as they operate with high degrees regio-, chemo-, stereoselectivity. 2-Oxoglutarate (2OG)-dependent nonheme iron halogenases chlorinate brominate aliphatic bonds offer a solution achieving these challenging transformations. Here, we describe the ability halogenase, SyrB2, to controllably halogenate non-native substrate α-aminobutyric acid (Aba) yield monochlorinated, dichlorinated, trichlorinated products. These chemoselective outcomes achieved by controlling loading 2OG cofactor SyrB2 biocatalyst. Furthermore, using ferredoxin-based biological reductant electron transfer catalytic center demonstrate order-of-magnitude enhancement were previously inaccessible any single halogenase enzyme. We also apply strategies broaden SyrB2's reactivity scope include multibromination chemoenzymatic conversion ethyl side chain Aba an ethynyl group. show how steric hindrance induced successive addition halogen atoms on Aba's C4 carbon dictates degree hampering C3–C4 bond rotation within pocket. Overall, our work showcases potential facilitate multi–C–H chemistry.

Language: Английский

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Synergistic Binding of the Halide and Cationic Prime Substrate of l-Lysine 4-Chlorinase, BesD, in Both Ferrous and Ferryl States

Biochemistry, Journal Year: 2023, Volume and Issue: 62(16), P. 2480 - 2491

Published: Aug. 5, 2023

An aliphatic halogenase requires four substrates: 2-oxoglutarate (2OG), halide (Cl

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Machine learning guided rational design of a non-heme iron-based lysine dioxygenase improves its total turnover number

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: June 5, 2024

Highly selective C-H functionalization remains an ongoing challenge in organic synthetic methodologies. Biocatalysts are robust tools for achieving these difficult chemical transformations. Biocatalyst engineering has often required directed evolution or structure-based rational design campaigns to improve their activities. In recent years, machine learning been integrated into workflows the discovery of beneficial enzyme variants. this work, we combine a machine-learning algorithm with classical molecular dynamics simulations down select mutations non-heme iron-dependent lysine dioxygenase, LDO. This approach consistently resulted functional LDO mutants and circumvents need extensive study mutational activity before-hand. Our rationally designed single purified up 2-fold higher yields than WT displayed total turnover numbers (TTN). Combining five such pentamutant variant, LPNYI LDO, leads 40% improvement TTN (218±3) as compared (TTN = 160±2). Overall, work offers low-barrier those seeking synergize algorithms pre-existing protein strategies.

Language: Английский

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