RESUMO
Galactose oxidase (GOase) is a Cu-dependent metalloenzyme that catalyzes the oxidation of alcohols to aldehydes. An evolved GOase variant was recently shown to catalyze a desymmetrizing oxidation as the first enzymatic step in the biocatalytic synthesis of islatravir. Horseradish peroxidase (HRP) is required to activate the GOase, introducing cost and protein burden to the process. Herein we describe that complexes of earth-abundant Mn(iii) (e.g. Mn(OAc)3) can be used at low loadings (2 mol%) as small molecule alternatives to HRP, providing similar yields and purity profiles. While an induction period is observed when using Mn(OAc)3 as the activator, employment of alternative Mn(iii) sources, such as Mn(acac)3 and K3[Mn(C2O4)3], eliminates the induction period and provides higher conversions to product. We demonstrate that use of the Mn(OAc)3 additive is also compatible with subsequent biocatalytic steps in the islatravir-forming cascade. Finally, to exhibit the wider utility of Mn(OAc)3, we show that Mn(OAc)3 functions as a suitable activator for several commercially available variants of GOase with a series of alcohol substrates.
RESUMO
The emergence of new therapeutic modalities requires complementary tools for their efficient syntheses. Availability of methodologies for site-selective modification of biomolecules remains a long-standing challenge, given the inherent complexity and the presence of repeating residues that bear functional groups with similar reactivity profiles. We describe a bioconjugation strategy for modification of native peptides relying on high site selectivity conveyed by enzymes. We engineered penicillin G acylases to distinguish among free amino moieties of insulin (two at amino termini and an internal lysine) and manipulate cleavable phenylacetamide groups in a programmable manner to form protected insulin derivatives. This enables selective and specific chemical ligation to synthesize homogeneous bioconjugates, improving yield and purity compared to the existing methods, and generally opens avenues in the functionalization of native proteins to access biological probes or drugs.
Assuntos
Insulina , Penicilina Amidase , Peptídeos , Engenharia de Proteínas , Sequência de Aminoácidos , Humanos , Insulina/análogos & derivados , Insulina/biossíntese , Lisina/química , Penicilina Amidase/química , Penicilina Amidase/genética , Peptídeos/química , Peptídeos/genética , Engenharia de Proteínas/métodosRESUMO
We have conducted a site-specific saturation mutagenesis study of H181 and H184 of flavoprotein pentaerythritol tetranitrate reductase (PETN reductase) to probe the role of these residues in substrate binding and catalysis with a variety of α,ß-unsaturated alkenes. Single mutations at these residues were sufficient to dramatically increase the enantiopurity of products formed by reduction of 2-phenyl-1-nitropropene. In addition, many mutants exhibited a switch in reactivity to predominantly catalyse nitro reduction, as opposed to CC reduction. These mutants showed an enhancement in a minor side reaction and formed 2-phenylpropanal oxime from 2-phenyl-1-nitropropene. The multiple binding conformations of hydroxy substituted nitro-olefins in PETN reductase were examined by using both structural and catalytic techniques. These compounds were found to bind in both active and inhibitory complexes; this highlights the plasticity of the active site and the ability of the H181/H184 couple to coordinate with multiple functional groups. These properties demonstrate the potential to use PETN reductase as a scaffold in the development of industrially useful biocatalysts.
Assuntos
Enterobacter cloacae/enzimologia , Mutagênese Sítio-Dirigida , Oxirredutases/genética , Oxirredutases/metabolismo , Shewanella/enzimologia , Aldeídos/metabolismo , Alcenos/metabolismo , Cristalografia por Raios X , Enterobacter cloacae/química , Enterobacter cloacae/genética , Modelos Moleculares , Oxirredutases/química , Oximas/metabolismo , Fenóis/metabolismo , Ligação Proteica , Shewanella/química , Shewanella/genética , Estereoisomerismo , Especificidade por SubstratoRESUMO
Molnupiravir (MK-4482) is an investigational antiviral agent that is under development for the treatment of COVID-19. Given the potential high demand and urgency for this compound, it was critical to develop a short and sustainable synthesis from simple raw materials that would minimize the time needed to manufacture and supply molnupiravir. The route reported here is enabled through the invention of a novel biocatalytic cascade featuring an engineered ribosyl-1-kinase and uridine phosphorylase. These engineered enzymes were deployed with a pyruvate-oxidase-enabled phosphate recycling strategy. Compared to the initial route, this synthesis of molnupiravir is 70% shorter and approximately 7-fold higher yielding. Looking forward, the biocatalytic approach to molnupiravir outlined here is anticipated to have broad applications for streamlining the synthesis of nucleosides in general.
RESUMO
This work describes the development of an automated robotic platform for the rapid screening of enzyme variants generated from directed evolution studies of pentraerythritol tetranitrate (PETN) reductase, a target for industrial biocatalysis. By using a 96-well format, near pure enzyme was recovered and was suitable for high throughput kinetic assays; this enabled rapid screening for improved and new activities from libraries of enzyme variants. Initial characterisation of several single site-saturation libraries targeted at active site residues of PETN reductase, are described. Two mutants (T26S and W102F) were shown to have switched in substrate enantiopreference against substrates (E)-2-aryl-1-nitropropene and α-methyl-trans-cinnamaldehyde, respectively, with an increase in ee (62 % (R) for W102F). In addition, the detection of mutants with weak activity against α,ß-unsaturated carboxylic acid substrates showed progress in the expansion of the substrate range of PETN reductase. These methods can readily be adapted for rapid evolution of enzyme variants with other oxidoreductase enzymes.
Assuntos
Proteínas de Bactérias/genética , Evolução Molecular Direcionada/métodos , Oxirredutases/genética , Biblioteca de Peptídeos , Acroleína/análogos & derivados , Acroleína/química , Acroleína/metabolismo , Alcenos/química , Alcenos/metabolismo , Anaerobiose , Bacillus subtilis/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Biocatálise , Ácidos Carboxílicos/química , Ácidos Carboxílicos/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Eletroforese em Gel de Poliacrilamida , Enterobacter cloacae/enzimologia , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , NADP/química , NADP/metabolismo , Oxirredutases/química , Oxirredutases/metabolismo , Estrutura Terciária de Proteína , Estereoisomerismo , Especificidade por SubstratoRESUMO
We report the crystal structure of a thermophilic "ene" reductase (TOYE) isolated from Thermoanaerobacter pseudethanolicus E39. The crystal structure reveals a tetrameric enzyme and an active site that is relatively large compared to most other structurally determined and related Old Yellow Enzymes. The enzyme adopts higher order oligomeric states (octamers and dodecamers) in solution, as revealed by sedimentation velocity and multiangle laser light scattering. Bead modelling indicates that the solution structure is consistent with the basic tetrameric structure observed in crystallographic studies and electron microscopy. TOYE is stable at high temperatures (T(m)>70 degrees C) and shows increased resistance to denaturation in water-miscible organic solvents compared to the mesophilic Old Yellow Enzyme family member, pentaerythritol tetranitrate reductase. TOYE has typical ene-reductase properties of the Old Yellow Enzyme family. There is currently major interest in using Old Yellow Enzyme family members in the preparative biocatalysis of a number of activated alkenes. The increased stability of TOYE in organic solvents is advantageous for biotransformations in which water-miscible organic solvents and biphasic reaction conditions are required to both deliver novel substrates and minimize product racemisation.
Assuntos
NADPH Desidrogenase/metabolismo , Alcenos/química , Biocatálise , Domínio Catalítico , Cristalografia por Raios X , Cinética , NADPH Desidrogenase/antagonistas & inibidores , NADPH Desidrogenase/genética , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Solventes/química , Estereoisomerismo , Especificidade por Substrato , Thermoanaerobacter/enzimologiaRESUMO
A short chemoenzymatic synthesis of beta-aryl-gamma-aminobutyric acids has been developed, based on a highly enantioselective biocatalytic reduction of beta-aryl-beta-cyano-alpha,beta-unsaturated carboxylic acids.
Assuntos
Bactérias/enzimologia , Oxirredutases/metabolismo , Ácido gama-Aminobutírico/biossíntese , Ácido gama-Aminobutírico/síntese química , Anaerobiose , Biocatálise , Oxirredução , Estereoisomerismo , Especificidade por Substrato , Ácido gama-Aminobutírico/análogos & derivados , Ácido gama-Aminobutírico/químicaRESUMO
Enzyme catalysis, enabled by advances in protein engineering and directed evolution, is beginning to transform chemical synthesis in the pharmaceutical industry. This review presents recent examples of the creative use of biocatalysis to enable drug discovery and development. We illustrate how increased access to novel biotransformations and the rise of cascade biocatalysis allowed fundamentally new syntheses of novel medicines, representing progress toward more sustainable pharmaceutical manufacturing. Finally, we describe the opportunities and challenges the industry must address to ensure the reduction to practice of biotechnological innovations to develop new therapies in a faster, more economical, and environmentally benign way.
Assuntos
Biocatálise , Química Farmacêutica/métodos , Enzimas/metabolismo , Alquilação , Biotransformação , Descoberta de Drogas , Indústria Farmacêutica , Indóis/síntese química , Metais/química , Oxirredução , Peptídeos/síntese química , Processos Fotoquímicos , Engenharia de Proteínas , Pirimidinas/síntese química , Pirróis/síntese química , Estereoisomerismo , Elementos de Transição/químicaRESUMO
The synthesis of the potent anti-HIV investigational treatment islatravir is described. The key step in this synthesis is a highly enantioselective catalytic asymmetric alkynylation of a ketone. This reaction is a rare example of the asymmetric addition of an alkyne nucleophile to a ketone through ligand-accelerated catalysis that was performed on a greater than 100 g scale. By leveraging a multienzyme cascade, a highly diastereoselective aldol-glycosylation was used to complete the target in eight steps.
RESUMO
We show that pentaerythritol tetranitrate reductase (PETNR), a member of the 'ene' reductase old yellow enzyme family, catalyses the asymmetric reduction of a variety of industrially relevant activated alpha,beta-unsaturated alkenes including enones, enals, maleimides and nitroalkenes. We have rationalised the broad substrate specificity and stereochemical outcome of these reductions by reference to molecular models of enzyme-substrate complexes based on the crystal complex of the PETNR with 2-cyclohexenone 4a. The optical purity of products is variable (49-99% ee), depending on the substrate type and nature of substituents. Generally, high enantioselectivity was observed for reaction products with stereogenic centres at Cbeta (>99% ee). However, for the substrates existing in two isomeric forms (e.g., citral 11a or nitroalkenes 18-19a), an enantiodivergent course of the reduction of E/Z-forms may lead to lower enantiopurities of the products. We also demonstrate that the poor optical purity obtained for products with stereogenic centres at Calpha is due to non-enzymatic racemisation. In reactions with ketoisophorone 3a we show that product racemisation is prevented through reaction optimisation, specifically by shortening reaction time and through control of solution pH. We suggest this as a general strategy for improved recovery of optically pure products with other biocatalytic conversions where there is potential for product racemisation.
RESUMO
Enzyme-catalyzed reactions have begun to transform pharmaceutical manufacturing, offering levels of selectivity and tunability that can dramatically improve chemical synthesis. Combining enzymatic reactions into multistep biocatalytic cascades brings additional benefits. Cascades avoid the waste generated by purification of intermediates. They also allow reactions to be linked together to overcome an unfavorable equilibrium or avoid the accumulation of unstable or inhibitory intermediates. We report an in vitro biocatalytic cascade synthesis of the investigational HIV treatment islatravir. Five enzymes were engineered through directed evolution to act on non-natural substrates. These were combined with four auxiliary enzymes to construct islatravir from simple building blocks in a three-step biocatalytic cascade. The overall synthesis requires fewer than half the number of steps of the previously reported routes.
Assuntos
Biocatálise , Desoxiadenosinas/química , Inibidores da Transcriptase Reversa/química , Biotecnologia/métodos , Preparações Farmacêuticas/síntese química , EstereoisomerismoRESUMO
Biocatalytic reduction of alpha- or beta-alkyl-beta-arylnitroalkenes provides a convenient and efficient method to prepare chiral substituted nitroalkanes. Pentaerythritol tetranitrate reductase (PETN reductase) from Enterobacter cloacae st. PB2 catalyses the reduction of nitroolefins such as 1-nitrocyclohexene (1) with steady state and rapid reaction kinetics comparable to other old yellow enzyme homologues. Furthermore, it reduces 2-aryl-1-nitropropenes (4a-d) to their equivalent (S)-nitropropanes 9a-d. The enzyme shows a preference for the (Z)-isomer of substrates 4a-d, providing almost pure enantiomeric products 9a-d (ees up to > 99%) in quantitative yield, whereas the respective (E)-isomers are reduced with lower enantioselectivity (63-89% ee) and lower product yields. 1-Aryl-2-nitropropenes (5a, b) are also reduced efficiently, but the products (R)-10 have lower optical purities. The structure of the enzyme complex with 1-nitrocyclohexene (1) was determined by X-ray crystallography, revealing two substrate-binding modes, with only one compatible with hydride transfer. Models of nitropropenes 4 and 5 in the active site of PETN reductase predicted that the enantioselectivity of the reaction was dependent on the orientation of binding of the (E)- and (Z)-substrates. This work provides a structural basis for understanding the mechanism of asymmetric bioreduction of nitroalkenes by PETN reductase.
RESUMO
Enzymes are natural catalysts, controlling reactions with typically high stereospecificity and enantiospecificity in substrate selection and/or product formation. This makes them useful in the synthesis of industrially relevant compounds, particularly where highly enantiopure products are required. The flavoprotein pentaerythritol tetranitrate (PETN) reductase is a member of the Old Yellow Enzyme family, and catalyses the asymmetric reduction of ß-alkyl-ß-arylnitroalkenes. Under aerobic conditions, it additionally undergoes futile cycles of NAD(P)H reduction of flavin, followed by reoxidation by oxygen, which generates the reactive oxygen species (ROS) hydrogen peroxide and superoxide. Prior studies have shown that not all reactions catalysed by PETN reductase yield enantiopure products, such as the reduction of (E)-2-phenyl-1-nitroprop-1-ene (PNE) to produce (S)-2-phenyl-1-nitropropane (PNA) with variable enantiomeric excess (ee). Recent independent studies of (E)-PNE reduction by PETN reductase showed that the major product formed could be switched to (R)-PNA, depending on the reaction conditions. We investigated this phenomenon, and found that the presence of oxygen and ROS influenced the overall product enantiopurity. Anaerobic reactions produced consistently higher nitroalkane (S)-PNA product yields than aerobic reactions (64% versus 28%). The presence of oxygen dramatically increased the preference for (R)-PNA formation (up to 52% ee). Conversely, the presence of the ROS superoxide and hydrogen peroxide switched the preference to (S)-PNA product formation. Given that oxygen has no role in the natural catalytic cycle, these findings demonstrate a remarkable ability to manipulate product enantiopurity of this enzyme-catalysed reaction by simple manipulation of reaction conditions. Potential mechanisms of this unusual behaviour are discussed.
Assuntos
Cicloparafinas/química , Cicloparafinas/metabolismo , Oxirredutases/metabolismo , Oxigênio/metabolismo , Catálise , Cisteína/metabolismo , Peróxido de Hidrogênio/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Metionina/metabolismo , NADP/metabolismo , Nitroparafinas/metabolismo , Propano/análogos & derivados , Propano/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Estereoisomerismo , Superóxidos/metabolismoRESUMO
This is the first report of the use of Clostridium sporogenes extracts for enantioselective reduction of CC double bonds of beta,beta-disubstituted (1) and alpha,beta-disubstituted nitroalkenes (3). Crude enzyme preparations reduced aryl derivatives 1a-e and 1h, in 35-86% yield with > or =97% ee. Reduction of (E)- and (Z)-isomers of 1c gave the same enantiomer of 2c (> or =99% ee). In contrast, alpha,beta-disubstituted nitroalkene 3a was a poor substrate, yielding (S)- 4a in low yield (10-20%), and the ee (30-70% ee) depended on NADH concentration. An efficient synthesis of a library of nitroalkenes 1 is described.
Assuntos
Alcenos/metabolismo , Clostridium/metabolismo , Biotransformação , Catálise , Espectroscopia de Ressonância Magnética , Oxirredução , Espectrometria de Massas por Ionização por Electrospray , EstereoisomerismoRESUMO
Four substituted 4H-benzo[1,2,4]thiadiazines 2 were prepared by condensation of the appropriate anilines and benzonitriles followed by oxidative cyclization. The preparation of three fluorinated derivatives 2b-2d proceeded smoothly, while the synthesis of 2a was problematic, presumably due to the relatively high electron density of the benzene ring. The four-ring derivatives 2c and 2d exhibited liquid crystalline properties (2c: Cr 95 SmA 158 I and 2d: Cr 142 SmA 212 I). 4H-Benzo[1,2,4]thiadiazines 2 were oxidized with AgO to generate the corresponding persistent radicals 1 (g=2.0057). The stability of the radicals followed the order 1b approximately 1d>1c>1a, and the two fluorinated radicals 1b and 1d were isolated as crude solids. The lower stability of 1c is presumably due to the presence of the reactive benzylic CH position, and 1a lacks the stabilizing effect of the three fluorine atoms. ESR spectra for 1 were simulated using DFT-derived hfcc as the starting point.