RESUMO
Herein, we report a one-pot, chemoenzymatic process for the synthesis of enantioenriched C(1)-allylated tetrahydroisoquinolines. This transformation couples a monoamine oxidase (MAO-N)-catalyzed oxidation with a metal catalyzed allylboration, followed by a biocatalytic deracemization to afford allylic amine derivatives in both high yields and good to high enantiomeric excess. The cascade is operationally simple, with all components added at the start of the reaction and can be used to generate key building blocks for further elaboration.
RESUMO
Organic chemistry provides society with fundamental products we use daily. Concerns about the impact that the chemical industry has over the environment is propelling major changes in the way we manufacture chemicals. Biocatalysis offers an alternative to other synthetic approaches as it employs enzymes, Nature's catalysts, to carry out chemical transformations. Enzymes are biodegradable, come from renewable sources, operate under mild reaction conditions, and display high selectivities in the processes they catalyse. As a highly multidisciplinary field, biocatalysis benefits from advances in different areas, and developments in the fields of molecular biology, bioinformatics, and chemical engineering have accelerated the extension of the range of available transformations (E.â L. Bell etâ al., Nat. Rev. Meth. Prim. 2021, 1, 1-21). Recently, we surveyed advances in the expansion of the scope of biocatalysis via enzyme discovery and protein engineering (J.â R. Marshall etâ al., Tetrahedron 2021, 82, 131926). Herein, we focus on novel enzymes currently available to the broad synthetic community for the construction of new C-C, C-N and C-O bonds, with the purpose of providing the non-specialist with new and alternative tools for chiral and sustainable chemical synthesis.
Assuntos
Enzimas , Engenharia de Proteínas , Biocatálise , Catálise , Enzimas/metabolismoRESUMO
The combination of small-molecule catalysis and enzyme catalysis represents an underexploited area of research with huge potential in asymmetric synthetic chemistry due to both compatibility of reaction conditions and complementary reactivity. Herein, we describe the telescopic synthesis of chiral nitro alcohols starting from commercially available benzaldehyde derivatives through the one-pot three-step chemoenzymatic cascade combination of a Wittig reaction, chiral-thiourea-catalysed asymmetric conjugate addition, and ketoreductase-mediated reduction to access the corresponding target compounds in moderate to excellent overall isolated yields (36-80 %) and high diastereomeric and enantiomeric ratios (up to >97 : 3). This represents the first example of the combination of an organocatalysed asymmetric conjugate addition via iminium ion activation and a bioreduction step catalysed by ketoreductases.
Assuntos
Álcoois , Benzaldeídos , Álcoois/química , Amino Álcoois , Biocatálise , Catálise , Estereoisomerismo , TioureiaRESUMO
OBJECTIVES: Formate dehydrogenases (FDHs) are NAD(P)H-dependent enzymes that catalyse the reversible oxidation of formate to CO2. The main goal was to use directed evolution to obtain variants of the FDH from Chaetomium thermophilum (CtFDH) with enhanced reduction activity in the conversion of CO2 into formic acid. RESULTS: Four libraries were constructed targeting five residues in the active site. We identified two variants (G93H/I94Y and R259C) with enhanced reduction activity which were characterised in the presence of both aqueous CO2(g) and HCO3-. The A1 variant (G93H/I94Y) showed a 5.4-fold increase in catalytic efficiency (kcat/KM) compared to that of the wild-type for HCO3- reduction. The improved biocatalysts were also applied as a coupled cofactor recycling system in the enantioselective oxidation of 4-phenyl-2-propanol catalysed by the alcohol dehydrogenase from Streptomyces coelicolor A3 (ScADH). Conversions in these reactions increased from 56 to 91% when the A1 variant was used instead of wild-type CtFDH. CONCLUSIONS: Two variants presenting up to five-fold increase in catalytic efficiency and kcat were obtained and characterised. They constitute a promising enzymatic alternative for CO2 utilization and will serve as scaffolds to be further developed in order to meet industrial requirements.
Assuntos
Dióxido de Carbono/metabolismo , Chaetomium/enzimologia , Formiato Desidrogenases/genética , Formiato Desidrogenases/metabolismo , Mutação , Álcool Desidrogenase/metabolismo , Biocatálise , Domínio Catalítico , Chaetomium/genética , Evolução Molecular Direcionada , Formiato Desidrogenases/química , Formiatos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Oxirredução , Propanóis/metabolismo , Engenharia de Proteínas , Streptomyces coelicolor/enzimologiaRESUMO
The alkylation of amines with either alcohols or carboxylic acids represents a mild and safe alternative to the use of genotoxic alkyl halides and sulfonate esters. Here we report two complementary one-pot systems in which the reductive aminase (RedAm) from Aspergillus oryzae is combined with either (i) a 1° alcohol/alcohol oxidase (AO) or (ii) carboxylic acid/carboxylic acid reductase (CAR) to affect N-alkylation reactions. The application of both approaches has been exemplified with respect to substrate scope and also preparative scale synthesis. These new biocatalytic methods address issues facing alternative traditional synthetic protocols such as harsh conditions, overalkylation and complicated workup procedures.
Assuntos
Álcoois/química , Aminas/síntese química , Ácidos Carboxílicos/química , Oxirredutases atuantes sobre Doadores de Grupo CH-NH2/química , Oxirredutases do Álcool/química , Alquilação , Aspergillus oryzae/enzimologia , Biocatálise , Estrutura Molecular , Oxirredutases/químicaRESUMO
Ene-reductases (EREDs) catalyze the reduction of electron-deficient CâC bonds. Herein, we report the first example of ERED-catalyzed net reduction of CâC bonds of enimines (α,ß-unsaturated imines). Preliminary studies suggest their hydrolyzed ring-open ω-amino enones are the likely substrates for this step. When combined with imine reductase (IRED)-mediated CâN reduction, the result is an efficient telescoped sequence for the preparation of diastereomerically enriched 2-substituted saturated amine heterocycles.
Assuntos
Biocatálise , Compostos Heterocíclicos/síntese química , Iminas/química , Oxirredutases/química , Compostos Heterocíclicos/química , Estrutura Molecular , Oxirredução , EstereoisomerismoRESUMO
Over the next decades, with the growing concern of rising atmospheric carbon dioxide (CO2) levels, the importance of investigating new approaches for its reduction becomes crucial. Reclamation of CO2 for conversion into biofuels represents an alternative and attractive production method that has been studied in recent years, now with enzymatic methods gaining more attention. Formate dehydrogenases (FDHs) are NAD(P)H-dependent oxidoreductases that catalyze the conversion of formate into CO2 and have been extensively used for cofactor recycling in chemoenzymatic processes. A new FDH from Clostridium ljungdahlii (ClFDH) has been recently shown to possess activity in the reverse reaction: the mineralization of CO2 into formate. In this study, we show the successful homologous expression of ClFDH in Escherichia coli. Biochemical and kinetic characterization of the enzyme revealed that this homologue also demonstrates activity toward CO2 reduction. Structural analysis of the enzyme through homology modeling is also presented.
Assuntos
Dióxido de Carbono/metabolismo , Clostridium/enzimologia , Formiato Desidrogenases/metabolismo , Formiatos/metabolismo , Sequência de Aminoácidos , Clostridium/química , Clostridium/metabolismo , Formiato Desidrogenases/química , Cinética , Metais/metabolismo , Modelos Moleculares , NAD/metabolismo , Oxirredução , Alinhamento de SequênciaRESUMO
Asymmetric reductive aminations are some of the most important reactions in the preparation of active pharmaceuticals, as chiral amines feature in many of the world's most important drugs. Although many enzymes have been applied to the synthesis of chiral amines, the development of reductive amination reactions that use enzymes is attractive, as it would permit the one-step transformation of readily available prochiral ketones into chiral amines of high optical purity. However, as most natural "reductive aminase" activities operate on keto acids, and many are able to use only ammonia as the amine donor, there is considerable scope for the engineering of natural enzymes for the reductive amination of ketones, and also for the preparation of secondary amines using alkylamines as donors. This review summarises research into the development of NAD(P)H-dependent dehydrogenases for the reductive amination of ketones, including amino acid dehydrogenases (AADHs), natural amine dehydrogenases (AmDHs), opine dehydrogenases (OpDHs) and imine reductases (IREDs). In each case knowledge of the structure and mechanism of the enzyme class is addressed, with a further description of the engineering of those enzymes for the reductive amination of ketones towards primary and also secondary amine products.
RESUMO
Here we describe a one-pot, three-enzyme, cascade involving a cytochrome P450 monooxygenase, an alcohol dehydrogenase and a reductive aminase for the synthesis of secondary amines from cycloalkanes. Amine product concentrations of up to 19.6 mM were achieved. The preparative scale amination of cyclohexane was also demonstrated with a space-time yield of 2 g L-1 d-1.
RESUMO
The reductive aminase from Aspergillus oryzae (AspRedAm) was combined with a single alcohol dehydrogenase (either metagenomic ADH-150, an ADH from Sphingobium yanoikuyae (SyADH), or a variant of the ADH from Thermoanaerobacter ethanolicus (TeSADH W110A)) in a redox-neutral cascade for the biocatalytic alkylation of amines using primary and secondary alcohols. Aliphatic and aromatic secondary amines were obtained in up to 99 % conversion, as well as chiral amines directly from the racemic alcohol precursors in up to >97 % ee, releasing water as the only byproduct.
RESUMO
Biocatalytic retrosynthetic analysis of dibenz[c,e]azepines has highlighted the use of imine reductase (IRED) and ω-transaminase (ω-TA) biocatalysts to establish the key stereocentres of these molecules. Several enantiocomplementary IREDs were identified for the synthesis of (R)- and (S)-5-methyl-6,7-dihydro-5H-dibenz[c,e]azepine with excellent enantioselectivity, by reduction of the parent imines. Crystallographic evidence suggests that IREDs may be able to bind one conformer of the imine substrate such that, upon reduction, the major product conformer is generated directly. ω-TA biocatalysts were also successfully employed for the production of enantiopure 1-(2-bromophenyl)ethan-1-amine, thus enabling an orthogonal route for the installation of chirality into dibenz[c,e]azepine framework.
Assuntos
Azepinas/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Transaminases/metabolismo , Azepinas/química , Biocatálise , Estrutura Molecular , EstereoisomerismoRESUMO
Pyridoxal-phosphate (PLP)-dependent enzymes catalyse a remarkable diversity of chemical reactions in nature. A1RDF1 from Arthrobacter aurescens TC1 is a fold typeâ I, PLP-dependent enzyme in the classâ III transaminase (TA) subgroup. Despite sharing 28 % sequence identity with its closest structural homologues, including ß-alanine:pyruvate and γ-aminobutyrate:α-ketoglutarate TAs, A1RDF1 displayed no TA activity. Activity screening revealed that the enzyme possesses phospholyase (E.C.â 4.2.3.2) activity towards O-phosphoethanolamine (PEtN), an activity described previously for vertebrate enzymes such as human AGXT2L1, enzymes for which no structure has yet been reported. In order to shed light on the distinctive features of PLP-dependent phospholyases, structures of A1RDF1 in complex with PLP (internal aldimine) and PLPâ PEtN (external aldimine) were determined, revealing the basis of substrate binding and the structural factors that distinguish the enzyme from classâ III homologues that display TA activity.
Assuntos
Transaminases/metabolismo , Arthrobacter/enzimologia , Sítios de Ligação , Biocatálise , Domínio Catalítico , Humanos , Simulação de Dinâmica Molecular , Fosfato de Piridoxal/química , Fosfato de Piridoxal/metabolismo , Transaminases/químicaRESUMO
Two complex structures of the γ-aminobutyrate (GABA) transaminase A1R958 from Arthrobacter aurescens TC1 are presented. The first, determined to a resolution of 2.80â Å, features the internal aldimine formed by reaction between the â-amino group of Lys295 and the cofactor pyridoxal phosphate (PLP); the second, determined to a resolution of 2.75â Å, features the external aldimine adduct formed between PLP and GABA in the first half-reaction. This is the first structure of a microbial GABA transaminase in complex with its natural external aldimine and reveals the molecular determinants of GABA binding in this enzyme.
Assuntos
4-Aminobutirato Transaminase/química , Arthrobacter/enzimologia , Fosfato de Piridoxal/química , 4-Aminobutirato Transaminase/genética , 4-Aminobutirato Transaminase/metabolismo , Arthrobacter/genética , Modelos Moleculares , Filogenia , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Fosfato de Piridoxal/metabolismo , Homologia Estrutural de ProteínaRESUMO
A simple and novel chemoenzymatic route has been applied for the first time in the synthesis of miconazole and econazole single enantiomers. Lipases and oxidoreductases have been tested in stereoselective processes; the best results were attained with oxidoreductases for the introduction of chirality in an adequate intermediate. The behaviors of a series of ketones and racemic alcohols in bioreductions and acetylation procedures, respectively, have been investigated; the best results were found with alcohol dehydrogenases A and T, which allowed the production of (R)-2-chloro-1-(2,4-dichlorophenyl)ethanol in enantiopure form under very mild reaction conditions. Final chemical modifications have been performed in order to isolate the target fungicides miconazole and econazole both as racemates and as single enantiomers. Biological evaluation of the racemates and single enantiomers has shown remarkable differences against the growth of several microorganisms; while (R)-miconazole seemed to account for most of the biological activity of racemic miconazole on all the strains tested, both enantiomers of econazole showed considerable biological activities. In this manner, (R)-econazole showed higher values against Candida krusei , while higher values were observed for (S)-econazole against Cryptococcus neoformans, Penicillium chrysogenum, and Aspergillus niger.
Assuntos
Antifúngicos/síntese química , Antifúngicos/farmacologia , Econazol/síntese química , Econazol/farmacologia , Hidrocarbonetos Clorados/química , Hidrocarbonetos Clorados/síntese química , Lipase/química , Miconazol/síntese química , Miconazol/farmacologia , Animais , Antifúngicos/química , Candida/química , Candida/efeitos dos fármacos , Econazol/química , Humanos , Espectroscopia de Ressonância Magnética , Miconazol/química , Testes de Sensibilidade Microbiana , Estrutura Molecular , EstereoisomerismoRESUMO
Finding faster and simpler ways to screen protein sequence space to enable the identification of new biocatalysts for asymmetric synthesis remains both a challenge and a rate-limiting step in enzyme discovery. Biocatalytic strategies for the synthesis of chiral amines are increasingly attractive and include enzymatic asymmetric reductive amination, which offers an efficient route to many of these high-value compounds. Here we report the discovery of over 300 new imine reductases and the production of a large (384 enzymes) and sequence-diverse panel of imine reductases available for screening. We also report the development of a facile high-throughput screen to interrogate their activity. Through this approach we identified imine reductase biocatalysts capable of accepting structurally demanding ketones and amines, which include the preparative synthesis of N-substituted ß-amino ester derivatives via a dynamic kinetic resolution process, with excellent yields and stereochemical purities.
Assuntos
Ensaios de Triagem em Larga Escala/métodos , Oxirredutases/isolamento & purificação , Aminação/efeitos dos fármacos , Aminas/química , Biocatálise , Iminas/metabolismo , Cetonas/química , Oxirredutases/metabolismo , EstereoisomerismoRESUMO
Chiral primary amines are important intermediates in the synthesis of pharmaceutical compounds. Fungal reductive aminases (RedAms) are NADPH-dependent dehydrogenases that catalyse reductive amination of a range of ketones with short-chain primary amines supplied in an equimolar ratio to give corresponding secondary amines. Herein we describe structural and biochemical characterisation as well as synthetic applications of two RedAms from Neosartorya spp. (NfRedAm and NfisRedAm) that display a distinctive activity amongst fungal RedAms, namely a superior ability to use ammonia as the amine partner. Using these enzymes, we demonstrate the synthesis of a broad range of primary amines, with conversions up to >97% and excellent enantiomeric excess. Temperature dependent studies showed that these homologues also possess greater thermal stability compared to other enzymes within this family. Their synthetic applicability is further demonstrated by the production of several primary and secondary amines with turnover numbers (TN) up to 14 000 as well as continous flow reactions, obtaining chiral amines such as (R)-2-aminohexane in space time yields up to 8.1 g L-1 h-1. The remarkable features of NfRedAm and NfisRedAm highlight their potential for wider synthetic application as well as expanding the biocatalytic toolbox available for chiral amine synthesis.
RESUMO
The resolution of methyl (+/-)-3-hydroxypentanoate catalysed by Candida antarctica lipase B has been performed by using ammonia and benzyl amine as nucleophiles. In all cases, the lipase reacts faster with the R enantiomer of the ester, but when benzyl amine is used, the enantiomeric ratio is approximately three times as high as that measured for ammonia. The analysis of the molecular dynamics simulations carried out over the corresponding deacylation transition state analogues indicated specular binding modes between enantiomers that vary greatly upon the nucleophile used. For the case of ammonia, an intramolecular hydrogen bond between the beta-hydroxyl group and the protons of the nucleophile is established. However, the presence of the substituent in benzyl amine disrupts this interaction. Instead, the acyl chain binds to a more restrictive area of the protein where the higher number of contacts established with the side chains of Thr40, Gln157 and Ile189 have been identified as the reason for the higher enantioselectivity observed in the aminolysis reaction.
Assuntos
Amônia/química , Benzilaminas/química , Candida/enzimologia , Lipase/química , Valeratos/química , Biocatálise , Domínio Catalítico , Simulação por Computador , Proteínas Fúngicas , Ligação de Hidrogênio , Lipase/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , EstereoisomerismoRESUMO
A straightforward chemoenzymatic synthesis of enantiomerically pure rivastigmine has been efficiently carried out under mild reaction conditions, with Candida antarctica lipase B responsible for the stereoselective acetylation of the corresponding (R)-alcohol or amine. An exhaustive enzymatic study has been developed exploring the possibilities of carry out enzyme recycling, scaling up the enzymatic process and development of a dynamic kinetic resolution procedure for the production of adequate enantiomerically pure precursors of rivastigmine. Total chemoenzymatic synthesis of this pharmaceutical has been performed in good overall yield from commercially available 3-methoxyacetophenone.
Assuntos
Candida/enzimologia , Lipase/metabolismo , Fenilcarbamatos/síntese química , Catálise , Lipase/química , Estrutura Molecular , Fenilcarbamatos/química , Rivastigmina , EstereoisomerismoRESUMO
Synthesis of the chiral amine moiety is a key challenge for synthetic organic chemistry due to its prevalence in many biologically active molecules. Imine reductase and amine oxidase enzymes have enabled the biocatalytic synthesis of a host of chiral amine compounds. In this chapter, procedures for the synthesis of chiral amines using imine reductases (IREDs), the recently discovered IRED homologues reductive aminases, and amine oxidases (AOs) are described. Amine oxidases have been the subject of mutagenesis approaches for improvement of substrate scope. The high-throughput screening method for determining active variants in amine oxidase libraries is illustrated. Finally, in an approach which takes inspiration from nature, many enzymes can be combined with each other in cascade reactions. The incorporation of imine reductase and monoamine oxidase biocatalysts into several cascade reactions, both in vitro and in vivo (where the approach moves toward synthetic biology), is reported.
Assuntos
Aminas/metabolismo , Aminoidrolases/metabolismo , Bactérias/enzimologia , Fungos/enzimologia , Monoaminoxidase/metabolismo , Oxirredutases/metabolismo , Engenharia de Proteínas/métodos , Aminas/química , Aminoidrolases/genética , Aspergillus niger/enzimologia , Aspergillus niger/genética , Aspergillus niger/metabolismo , Bactérias/genética , Bactérias/metabolismo , Biocatálise , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Fungos/genética , Fungos/metabolismo , Iminas/química , Iminas/metabolismo , Monoaminoxidase/genética , Oxirredução , Oxirredutases/genética , Estereoisomerismo , Streptomyces/enzimologia , Streptomyces/genética , Streptomyces/metabolismo , Biologia Sintética/métodosRESUMO
Reductive amination is one of the most important methods for the synthesis of chiral amines. Here we report the discovery of an NADP(H)-dependent reductive aminase from Aspergillus oryzae (AspRedAm, Uniprot code Q2TW47) that can catalyse the reductive coupling of a broad set of carbonyl compounds with a variety of primary and secondary amines with up to >98% conversion and with up to >98% enantiomeric excess. In cases where both carbonyl and amine show high reactivity, it is possible to employ a 1:1 ratio of the substrates, forming amine products with up to 94% conversion. Steady-state kinetic studies establish that the enzyme is capable of catalysing imine formation as well as reduction. Crystal structures of AspRedAm in complex with NADP(H) and also with both NADP(H) and the pharmaceutical ingredient (R)-rasagiline are reported. We also demonstrate preparative scale reductive aminations with wild-type and Q240A variant biocatalysts displaying total turnover numbers of up to 32,000 and space time yields up to 3.73â gâ l-1â d-1.