RESUMEN
Fluorinated cyclopropanes are highly desired pharmacophores in drug discovery owing to the rigid nature of the cyclopropane ring and the beneficial effects of C-F bonds on the pharmacokinetic properties, cell permeability, and metabolic stability of drug molecules. Herein a biocatalytic strategy for the stereoselective synthesis of mono-fluorinated and gem-difluoro cyclopropanes is reported though the use of engineered myoglobin-based catalysts. In particular, this system allows for a broad range of gem-difluoro alkenes to be cyclopropanated in the presence of diazoacetonitrile with excellent diastereo and enantiocontrol (up to 99 : 1â d.r. and 99 % e.e.), thereby enabling a transformation not currently accessible with chemocatalytic methods. The synthetic utility of the present approach is further exemplified through the gram-scale synthesis of a key gem-difluorinated cyclopropane intermediate useful for the preparation of fluorinated bioactive molecules.
RESUMEN
The exploration of large DNA libraries of metagenomic or synthetic origin is greatly facilitated by ultrahigh-throughput assays that use monodisperse water-in-oil emulsion droplets as sequestered reaction compartments. Millions of samples can be generated and analysed in microfluidic devices at kHz speeds, requiring only micrograms of reagents. The scope of this powerful platform for the discovery of new sequence space is, however, hampered by the limited availability of assay substrates, restricting the functions and reaction types that can be investigated. Here, we broaden the scope of detectable biochemical transformations in droplet microfluidics by introducing the first fluorogenic assay for alcohol dehydrogenases (ADHs) in this format. We have synthesized substrates that release a pyranine fluorophore (8-hydroxy-1,3,6-pyrenetrisulfonic acid, HPTS) when enzymatic turnover occurs. Pyranine is well retained in droplets for >6 weeks (i. e. 14-times longer than fluorescein), avoiding product leakage and ensuring excellent assay sensitivity. Product concentrations as low as 100â nM were successfully detected, corresponding to less than one turnover per enzyme molecule on average. The potential of our substrate design was demonstrated by efficient recovery of a bona fide ADH with an >800-fold enrichment. The repertoire of droplet screening is enlarged by this sensitive and direct fluorogenic assay to identify dehydrogenases for biocatalytic applications.
Asunto(s)
Alcohol Deshidrogenasa/análisis , Colorantes Fluorescentes/química , Ensayos Analíticos de Alto Rendimiento , Dispositivos Laboratorio en un Chip , Alcohol Deshidrogenasa/metabolismo , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/metabolismo , Estructura Molecular , Tamaño de la PartículaRESUMEN
Cytochrome P450 BM3 monooxygenases are able to catalyze the regio- and stereoselective oxygenation of a broad range of substrates, with promising potential for synthetic applications. To study the suitability of P450 BM3 variants for stereoselective benzylic hydroxylation of 2-alkylated benzoic acid esters, the biotransformation of methyl 2-ethylbenzoate, resulting in both enantiomeric forms of 3-methylphthalide, was investigated. In the case of methyl 2-propylbenzoate as a substrate the regioselectivity of the reaction was shifted towards ß-hydroxylation, resulting in the synthesis of enantioenriched R- and S-configured 3-methylisochroman-1-one. The potential of P450 BM3 variants for regio- and stereoselective synthesis of phthalides and isocoumarins offers a new route to a class of compounds that are valuable synthons for a variety of natural compounds.
Asunto(s)
Proteínas Bacterianas/química , Benzofuranos/síntesis química , Sistema Enzimático del Citocromo P-450/química , Isocumarinas/síntesis química , Lactonas/síntesis química , NADPH-Ferrihemoproteína Reductasa/química , Sustitución de Aminoácidos , Bacillus megaterium , Hidroxilación , Ingeniería de Proteínas , EstereoisomerismoRESUMEN
Fluorescence-based assays for the cytochrome P450 BM3 monooxygenase from Bacillus megaterium address an attractive biotechnological challenge by facilitating enzyme engineering and the identification of potential substrates of this highly promising biocatalyst. In the current study, we used the scarcity of corresponding screening systems as an opportunity to evaluate a novel and continuous high-throughput assay for this unique enzyme. A set of nine catalytically diverse P450 BM3 variants was constructed and tested toward the native substrate-inspired fluorogenic substrate 12-(4-trifluoromethylcoumarin-7-yloxy)dodecanoic acid. Particularly high enzyme-mediated O-dealkylation yielding the fluorescent product 7-hydroxy-4-trifluoromethylcoumarin was observed with mutants containing the F87V substitution, with A74G/F87V showing the highest catalytic efficiency (0.458 min(-1)µM(-1)). To simplify the assay procedure and show its versatility, different modes of application were successfully demonstrated, including (i) the direct use of NADPH or its oxidized form NADP(+) along with diverse NADPH recycling systems for electron supply, (ii) the use of cell-free lysates and whole-cell preparations as the biocatalyst source, and (iii) its use for competitive inhibition screens to identify or characterize substrates and inhibitors. A detailed comparison with known, fluorescence-based P450 BM3 assays finally emphasizes the relevance of our contribution to the ongoing research.
Asunto(s)
Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/metabolismo , Cumarinas/química , Cumarinas/metabolismo , Inhibidores Enzimáticos del Citocromo P-450/farmacología , Sistema Enzimático del Citocromo P-450/metabolismo , Evaluación Preclínica de Medicamentos/métodos , NADPH-Ferrihemoproteína Reductasa/antagonistas & inhibidores , NADPH-Ferrihemoproteína Reductasa/metabolismo , Alquilación , Biocatálisis , Cumarinas/síntesis química , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/química , Colorantes Fluorescentes/metabolismo , Ensayos Analíticos de Alto Rendimiento , Hidroxilación , Concentración 50 Inhibidora , Cinética , NADP/metabolismo , Oxazinas/química , Oxazinas/metabolismoRESUMEN
Chiral allylic alcohols of ω-alkenoic acids and derivatives thereof are highly important building blocks for the synthesis of biologically active compounds. The direct enantioselective C-H oxidation of linear terminal olefins offers the shortest route toward these compounds, but known synthetic methods are limited and suffer from low selectivities. Described herein is an enzymatic approach using the P450 BM3 monooxygenase mutant A74G/L188Q, which catalyzes allylic hydroxylation with high to excellent chemo- and enantioselectivities providing the desirable secondary alcohols.
Asunto(s)
Alquenos/química , Ésteres/química , Catálisis , Hidroxilación , Oxidación-Reducción , EstereoisomerismoRESUMEN
The success of protein evolution campaigns is strongly dependent on the sequence context in which mutations are introduced, stemming from pervasive non-additive interactions between a protein's amino acids ('intra-gene epistasis'). Our limited understanding of such epistasis hinders the correct prediction of the functional contributions and adaptive potential of mutations. Here we present a straightforward unique molecular identifier (UMI)-linked consensus sequencing workflow (UMIC-seq) that simplifies mapping of evolutionary trajectories based on full-length sequences. Attaching UMIs to gene variants allows accurate consensus generation for closely related genes with nanopore sequencing. We exemplify the utility of this approach by reconstructing the artificial phylogeny emerging in three rounds of directed evolution of an amine dehydrogenase biocatalyst via ultrahigh throughput droplet screening. Uniquely, we are able to identify lineages and their founding variant, as well as non-additive interactions between mutations within a full gene showing sign epistasis. Access to deep and accurate long reads will facilitate prediction of key beneficial mutations and adaptive potential based on in silico analysis of large sequence datasets.
Asunto(s)
Evolución Molecular Dirigida , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , Ingeniería de Proteínas/métodos , Biocatálisis , Clonación Molecular , Biología Computacional/métodos , Secuencia de Consenso/genética , Conjuntos de Datos como Asunto , Pruebas de Enzimas , Epistasis Genética , Biblioteca de Genes , Mutagénesis , Mutación , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/aislamiento & purificación , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Filogenia , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Programas InformáticosRESUMEN
The use of enzymes in organic solvents offers a great opportunity for the highly selective synthesis of complex organic compounds. In this study we investigate the POXylation of several enzymes with different polyoxazolines ranging from the hydrophilic poly(2-methyl-oxazoline) (PMOx) to the hydrophobic poly(2-heptyl-oxazoline) (PHeptOx). As reported previously on the examples of model enzymes POXylation mediated by pyromellitic acid dianhydride results in highly modified, organosoluble protein conjugates. This procedure is here extended to a larger number of proteins and optimized for the different polyoxazolines. The resulting polymer-enzyme conjugates (PEC) became soluble in different organic solvents ranging from hydrophilic DMF to even toluene. These conjugates were characterized regarding their solubility and especially their activity in organic solvents and in some cases the PECs showed significantly (up to 153,000 fold) higher activities than the respective native enzymes.