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The fortieth anniversary of biocatalysis started at Ciba-Geigy and later at Novartis is a great time to pause and reflect on development of science and technology in this field. Enzyme-based synthesis became a highly valued enabling tool for pharmaceutical research and development over the last decades. In this perspective we aim to discuss how the scientific approaches and trends evolved over the time and present future challenges and opportunities.
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BiocatálisisRESUMEN
We present a short overview of the way Novartis chemists interact and collaborate with the academic chemistry community in Switzerland. This article exemplifies a number of collaborations, and illustrates opportunities to foster research synergies between academic and industrial researchers. It also describes established programs available to academic groups, providing them access to Novartis resources and expertise.
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Industrias , Investigadores , Humanos , SuizaRESUMEN
Biocatalysis has found numerous applications in various fields as an alternative to chemical catalysis. The use of enzymes in organic synthesis, especially to make chiral compounds for pharmaceuticals as well for the flavors and fragrance industry, are the most prominent examples. In addition, biocatalysts are used on a large scale to make specialty and even bulk chemicals. This review intends to give illustrative examples in this field with a special focus on scalable chemical production using enzymes. It also discusses the opportunities and limitations of enzymatic syntheses using distinct examples and provides an outlook on emerging enzyme classes.
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Biocatálisis , Biotecnología/métodos , Enzimas/metabolismoRESUMEN
Enzymes are versatile biocatalysts capable of performing selective reactions. The advantages of enzymes in comparison to classical chemistry including chemical catalysts are the generally milder process conditions and avoidance of harmful reactants. Their high selectivity and specificity are especially beneficial for the enzymatic synthesis of new products with potential applications in drug research. Therefore, in the past decades, the utilization of isolated enzymes or whole-cell biocatalysts has spread through a growing number of biotechnological industries. The applications comprise the production of chiral building blocks for the pharmaceutical and fine chemical industry, the enzymatic synthesis of drug metabolites for testing of toxicity, function, biological activity, degradation and the production of biocatalytically modified natural products, which all play a role in drug discovery. Especially Oreste Ghisalba's contributions, which paved the way for the industrial use of enzymes, will be considered in this review.
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Descubrimiento de Drogas , Biocatálisis , Productos Biológicos , Biotecnología , EnzimasRESUMEN
Non-heme iron halogenases are synthetically valuable biocatalysts that are capable of halogenating unactivated sp3 -hybridized carbon centers with high stereo- and regioselectivity. The reported substrate scope of these enzymes, however, is limited primarily to the natural substrates and their analogues. We engineered the halogenase WelO5* for chlorination of a martinelline-derived fragment. Using structure-guided evolution, a halogenase variant with a more than 290-fold higher total turnover number and a 400-fold higher apparent kcat compared to the wildtype enzyme was generated. Moreover, we identified key positions in the active site that allow direction of the halogen to different positions in the target substrate. This is the first example of enzyme engineering to expand the substrate scope of a non-heme iron halogenase beyond the native indole-alkaloid-type substrates. The highly evolvable nature of WelO5* underscores the usefulness of this enzyme family for late-stage halogenation.
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Proteínas Bacterianas/metabolismo , Halogenación/genética , Humanos , Estructura Molecular , Especificidad por SustratoRESUMEN
A one-pot, two-step biocatalytic platform for the regiospecfic C-methylation and C-ethylation of aromatic substrates is described. The tandem process utilises SalL (Salinospora tropica) for in situ synthesis of S-adenosyl-l-methionine (SAM), followed by alkylation of aromatic substrates by the C-methyltransferase NovO (Streptomyces spheroides). The application of this methodology is demonstrated for the regiospecific labelling of aromatic substrates by the transfer of methyl, ethyl and isotopically labelled 13 CH3,13 CD3 and CD3 groups from their corresponding SAM analogues formed in situ.
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Metiltransferasas/metabolismo , S-Adenosilmetionina/metabolismo , Alquilación , Organismos Acuáticos , Bacterias/enzimología , Bacterias/metabolismo , Biocatálisis , Compuestos Heterocíclicos de Anillos Fusionados/metabolismo , Hidrocarburos Aromáticos/metabolismo , Marcaje Isotópico , MetilaciónRESUMEN
Enzymes have firmly established themselves as bespoke catalysts for small molecule transformations in the pharmaceutical industry, from early research and development stages to large-scale production. In principle, their exquisite selectivity and rate acceleration can also be leveraged for modifying macromolecules to form bioconjugates. However, available catalysts face stiff competition from other bioorthogonal chemistries. In this Perspective, we seek to illuminate applications of enzymatic bioconjugation in the face of an expanding palette of new drug modalities. With these applications, we wish to highlight some examples of current successes and pitfalls of using enzymes for bioconjugation along the pipeline and try to illustrate opportunities for further development.
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We have developed a chiral route toward the synthesis of muscarinic M4 agonists that was enabled by the biocatalytic synthesis of the key spirocyclic diamine building blocks 10 and 12. Using these bifunctional compounds we were able to optimize a synthetic sequence toward a collection of advanced intermediates for further elaboration. These advanced intermediates were then used as starting points for early medicinal chemistry and the identification of selective M1/M4 agonists.
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Several Baeyer-Villiger monooxygenases converting a wide spectrum of substrates have been discovered, cloned, and characterized throughout the last few years. Still, only a few of them are applicable for large-scale conversion predominantly due to their sensitivity towards high substrate and/or product concentrations. The recently cloned and characterized 4-hydroxyacetophenone monooxygenase from Pseudomonas putida JD1 shows excellent enantioselectivity towards 3-phenyl-2-butanone with E > 100 but is inhibited by concentrations >10 mM of both substrate and product. This obstacle could be circumvented by in situ substrate feed and product removal using a hydrophobic Lewatit® adsorbent resin. Thus, the concentration of 3-phenyl-2-butanone could be increased from 1.4 to >26 mM without significant reduction in conversion.
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Butanonas/metabolismo , Oxigenasas de Función Mixta/metabolismo , Oxigenasas/metabolismo , Pseudomonas putida/enzimología , Biocatálisis , Biotecnología , Butanonas/química , Escherichia coli/enzimología , Escherichia coli/genética , Escherichia coli/metabolismo , Microbiología Industrial/métodos , Redes y Vías Metabólicas , Oxigenasas de Función Mixta/química , NADP/metabolismo , Oxidación-Reducción , Pseudomonas putida/metabolismo , Resinas Sintéticas , Estereoisomerismo , Especificidad por SustratoRESUMEN
Chiral ß-hydroxy α-amino acid structural motifs are interesting and common synthons present in multiple APIs and drug candidates. To access these chiral building blocks either multistep chemical syntheses are required or the application of threonine aldolases, which catalyze aldol reactions between an aldehyde and glycine. Bioinformatics tools have been utilized to identify the gene encoding threonine aldolase from Vanrija humicola and subsequent preparation of its recombinant version from E. coli fermentation. We planned to implement this enzyme as a key step to access the synthesis of our target API. Beyond this specific application, the aldolase was purified, characterized and the substrate scope of this enzyme further investigated. A number of enzymatic reactions were scaled-up and the products recovered to assess the diastereoselectivity and scalability of this asymmetric synthetic approach towards ß-hydroxy α-amino acid chiral building blocks.
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Aminoácidos/metabolismo , Glicina Hidroximetiltransferasa/metabolismo , Aldehídos/química , Aminoácidos/química , Biocatálisis , Escherichia coli/metabolismo , Hongos/enzimología , Glicina Hidroximetiltransferasa/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Especificidad por SustratoRESUMEN
Multi-step enzyme reactions offer considerable cost and productivity benefits. Process models offer a route to understanding the complexity of these reactions, and allow for their optimization. Despite the increasing prevalence of multi-step biotransformations, there are few examples of process models for enzyme reactions. From a toolbox of characterized enzyme parts, we demonstrate the construction of a process model for a seven enzyme, three step biotransformation using isolated enzymes. Enzymes for cofactor regeneration were employed to make this inâ vitro reaction economical. Good modelling practice was critical in evaluating the impact of approximations and experimental error. We show that the use and validation of process models was instrumental in realizing and removing process bottlenecks, identifying divergent behavior, and for the optimization of the entire reaction using a genetic algorithm. We validated the optimized reaction to demonstrate that complex multi-step reactions with cofactor recycling involving at least seven enzymes can be reliably modelled and optimized.
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Recombinant Escherichia coli cells expressing eight Baeyer-Villiger monooxygenases of bacterial origin have been utilized to oxidize prochiral heterocyclic ketones containing a pyran ring system. Within the biotransformation, two stereogenic centers were introduced with high control of enantioselectivity. The chemoselectivity of the enzymatic reaction was found to be high in favor of the Baeyer-Villiger process when using substituted ketone precursors incorporating functional groups labile to oxidation. A significantly different behavior was observed for two groups of monooxygenases with respect to substrate acceptance, which is consistent with our previous classification into two enzyme clusters.
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Escherichia coli/enzimología , Cetonas/metabolismo , Oxigenasas de Función Mixta/metabolismo , Piranos/metabolismo , Proteínas Recombinantes/metabolismo , Biotransformación , Escherichia coli/genética , Cetonas/química , Oxigenasas de Función Mixta/química , Oxigenasas de Función Mixta/genética , Estructura Molecular , Oxidación-Reducción , Piranos/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , EstereoisomerismoRESUMEN
Carboxylic acid reductase enzymes (CARs) meet the demand in synthetic chemistry for a green and regiospecific route to aldehydes from their respective carboxylic acids. However, relatively few of these enzymes have been characterized. A sequence alignment with members of the ANL (Acyl-CoA synthetase/ NRPS adenylation domain/Luciferase) superfamily of enzymes shed light on CAR functional dynamics. Four unstudied enzymes were selected by using a phylogenetic analysis of known and hypothetical CARs, and for the first time, a thorough biochemical characterization was performed. Kinetic analysis of these enzymes with various substrates shows that they have a broad but similar substrate specificity. Electron-rich acids are favored, which suggests that the first step in the proposed reaction mechanism, attack by the carboxylate on the α-phosphate of adenosine triphosphate (ATP), is the step that determines the substrate specificity and reaction kinetics. The effects of pH and temperature provide a clear operational window for the use of these CARs, whereas an investigation of product inhibition by NADP+, adenosine monophosphate, and pyrophosphate indicates that the binding of substrates at the adenylation domain is ordered with ATP binding first. This study consolidates CARs as important and exciting enzymes in the toolbox for sustainable chemistry and provides specifications for their use as a biocatalyst.
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Escherichia coli/enzimología , Esterasas/química , Fenilbutiratos/química , Biocatálisis , Ácidos Carboxílicos/química , Ácidos Carboxílicos/metabolismo , Escherichia coli/citología , Escherichia coli/metabolismo , Esterasas/metabolismo , Citometría de Flujo , Glicerol/química , Glicerol/metabolismo , Cinética , Estructura Molecular , Fenilbutiratos/metabolismo , Propanoles/química , Propanoles/metabolismo , Estereoisomerismo , Especificidad por SustratoRESUMEN
P450tol monooxygenase was discovered as a unique and highly enantioselective enzyme for asymmetric epoxidation of some terminal alkenes containing electron-withdrawing groups and benzylic hydroxylation of several ethylbenzenes giving the corresponding useful and valuable products, such as (R)-2- and 3-substituted styrene oxides, (S)-4-substituted styrene oxides, and (S)-benzylic alcohols, in high ee.
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Alquenos/química , Derivados del Benceno/química , Compuestos Epoxi/química , Oxigenasas/química , Rhodococcus/enzimología , Escherichia coli/enzimología , Hidroxilación , Relación Estructura-Actividad , Estirenos/químicaAsunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/clasificación , Cetonas/metabolismo , Oxigenasas de Función Mixta/química , Oxigenasas de Función Mixta/clasificación , Secuencia de Aminoácidos , Biotransformación , Análisis por Conglomerados , Datos de Secuencia Molecular , Filogenia , Estereoisomerismo , Especificidad por SustratoRESUMEN
Bridged cycloketones were synthesized and utilized as substrates to study biooxidations mediated by Baeyer-Villiger monooxygenases (BVMO) of various bacterial origin. The required enzymes were heterologously produced by recombinant overexpression systems based on Escherichia coli to enable facile recycling of the required nicotinamide cofactors during the whole-cell biotransformations. Ketone precursors of various structural demands were chosen to evaluate steric limitations and flexibility of the active site of BVMOs. By desymmetrization of the prochiral substrates, four to six stereogenic centers were generated within a single biooxidation step. The enzyme library investigated in this study allowed access to antipodal lactone products with excellent enantioselectivity in several cases. Together with a distinct substrate acceptance profile, the recently proposed classification into two groups of cycloketone converting BVMOs was supported by the biotransformation results obtained within this study.
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Bacterias/enzimología , Cetonas/química , Oxigenasas de Función Mixta/química , Bacterias/genética , Catálisis , Ciclización , Escherichia coli/enzimología , Escherichia coli/genética , Cetonas/síntesis química , Cetonas/metabolismo , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Estructura Molecular , Oxidación-Reducción , Biblioteca de Péptidos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , EstereoisomerismoRESUMEN
Recombinant Escherichia coli B834 (DE3) pDB5 expressing the Rv3049c gene encoding a Baeyer-Villiger monooxygenase from Mycobacterium tuberculosis H37Rv was used for regioselective oxidations of fused bicyclic ketones. This whole-cell system represents the first recombinant Baeyer-Villiger oxidation biocatalyst that effectively resolves the racemic starting materials in this series. Within biotransformations using this organism one substrate enantiomer remains in high optical purity, while the second enantiomer is oxidized to one type of regioisomeric lactone preferably.