RESUMO
Chiral amine diastereomers are ubiquitous in pharmaceuticals and agrochemicals1, yet their preparation often relies on low-efficiency multi-step synthesis2. These valuable compounds must be manufactured asymmetrically, as their biochemical properties can differ based on the chirality of the molecule. Herein we characterize a multifunctional biocatalyst for amine synthesis, which operates using a mechanism that is, to our knowledge, previously unreported. This enzyme (EneIRED), identified within a metagenomic imine reductase (IRED) collection3 and originating from an unclassified Pseudomonas species, possesses an unusual active site architecture that facilitates amine-activated conjugate alkene reduction followed by reductive amination. This enzyme can couple a broad selection of α,ß-unsaturated carbonyls with amines for the efficient preparation of chiral amine diastereomers bearing up to three stereocentres. Mechanistic and structural studies have been carried out to delineate the order of individual steps catalysed by EneIRED, which have led to a proposal for the overall catalytic cycle. This work shows that the IRED family can serve as a platform for facilitating the discovery of further enzymatic activities for application in synthetic biology and organic synthesis.
Assuntos
Aminas , Oxirredutases , Aminação , Aminas/química , Biocatálise , Iminas/química , Oxirredutases/genética , Oxirredutases/metabolismo , EstereoisomerismoRESUMO
The development of efficient and sustainable methods for the synthesis of nitrogen heterocycles is an important goal for the chemical industry. In particular, substituted chiral piperidines are prominent targets due to their prevalence in medicinally relevant compounds and their precursors. A potential biocatalytic approach to the synthesis of this privileged scaffold would be the asymmetric dearomatization of readily assembled activated pyridines. However, nature is yet to yield a suitable biocatalyst specifically for this reaction. Here, by combining chemical synthesis and biocatalysis, we present a general chemo-enzymatic approach for the asymmetric dearomatization of activated pyridines for the preparation of substituted piperidines with precise stereochemistry. The key step involves a stereoselective one-pot amine oxidase/ene imine reductase cascade to convert N-substituted tetrahydropyridines to stereo-defined 3- and 3,4-substituted piperidines. This chemo-enzymatic approach has proved useful for key transformations in the syntheses of antipsychotic drugs Preclamol and OSU-6162, as well as for the preparation of two important intermediates in synthetic routes of the ovarian cancer monotherapeutic Niraparib.
Assuntos
Piperidinas , Piridinas , Piridinas/química , Estereoisomerismo , Catálise , Piperidinas/química , Iminas/químicaRESUMO
There has been growing interest in performing organocatalysis within a supramolecular system as a means of controlling reaction reactivity and stereoselectivity. Here, a protein is used as a host for iminium catalysis. A pyrrolidine moiety is covalently linked to biotin and introduced to the protein host streptavidin for organocatalytic activity. Whereas in traditional systems stereoselectivity is largely controlled by the substituents added to the organocatalyst, enantiomeric enrichment by the reported supramolecular system is completely controlled by the host. Also, the yield of the model reaction increases over 10-fold when streptavidin is included. A 1.1â Å crystal structure of the protein-catalyst complex and molecular simulations of a key intermediate reveal the chiral scaffold surrounding the organocatalytic reaction site. This work illustrates that proteins can be an excellent supramolecular host for driving stereoselective secondary amine organocatalysis.
Assuntos
Iminas/química , Estreptavidina/química , Sítios de Ligação , Biotina/química , Biotina/metabolismo , Biotinilação , Catálise , Cromatografia Gasosa-Espectrometria de Massas , Ligação de Hidrogênio , Conformação Molecular , Simulação de Dinâmica Molecular , Estereoisomerismo , Estreptavidina/metabolismoRESUMO
Mammalian dihydrofolate reductases (DHFRs) catalyze the reduction of folate more efficiently than the equivalent bacterial enzymes do, despite typically having similar efficiencies for the reduction of their natural substrate, dihydrofolate. In contrast, we show here that DHFR from the hyperthermophilic bacterium Thermotoga maritima can catalyze reduction of folate to tetrahydrofolate with an efficiency similar to that of reduction of dihydrofolate under saturating conditions. Nuclear magnetic resonance and mass spectrometry experiments showed no evidence of the production of free dihydrofolate during either the EcDHFR- or TmDHFR-catalyzed reductions of folate, suggesting that both enzymes perform the two reduction steps without release of the partially reduced substrate. Our results imply that the reaction proceeds more efficiently in TmDHFR than in EcDHFR because the more open active site of TmDHFR facilitates protonation of folate. Because T. maritima lives under extreme conditions where tetrahydrofolate is particularly prone to oxidation, this ability to salvage folate may impart an advantage to the bacterium by minimizing the squandering of a valuable cofactor.
Assuntos
Proteínas de Bactérias/química , Ácido Fólico/química , Prótons , Tetra-Hidrofolato Desidrogenase/química , Tetra-Hidrofolatos/química , Thermotoga maritima/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Escherichia coli/química , Escherichia coli/enzimologia , Escherichia coli/genética , Ácido Fólico/metabolismo , Expressão Gênica , Concentração de Íons de Hidrogênio , Cinética , NADP/química , NADP/metabolismo , Oxirredução , Dobramento de Proteína , Estrutura Secundária de Proteína , Especificidade da Espécie , Temperatura , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/metabolismo , Tetra-Hidrofolatos/metabolismo , Termodinâmica , Thermotoga maritima/química , Thermotoga maritima/genéticaRESUMO
Pterin-containing natural products have diverse functions in life, but an efficient and easy scheme for their in vitro synthesis is not available. Here we report a chemoenzymatic 14-step, one-pot synthesis that can be used to generate 13C- and 15N-labeled dihydrofolates (H2F) from glucose, guanine, and p-aminobenzoyl-l-glutamic acid. This synthesis stands out from previous approaches to produce H2F in that the average yield of each step is >91% and it requires only a single purification step. The use of a one-pot reaction allowed us to overcome potential problems with individual steps during the synthesis. The availability of labeled dihydrofolates allowed the measurement of heavy-atom isotope effects for the reaction catalyzed by the drug target dihydrofolate reductase and established that protonation at N5 of H2F and hydride transfer to C6 occur in a stepwise mechanism. This chemoenzymatic pterin synthesis can be applied to the efficient production of other folates and a range of other natural compounds with applications in nutritional, medical, and cell-biological research.
Assuntos
Ácido Fólico/biossíntese , Marcação por Isótopo , Tetra-Hidrofolato Desidrogenase/metabolismo , Isótopos de Carbono , Ácido Fólico/análogos & derivados , Ácido Fólico/química , Estrutura Molecular , Isótopos de Nitrogênio , Tetra-Hidrofolato Desidrogenase/químicaRESUMO
The potential of antibody conjugates with high drug loading in anticancer therapy has recently been highlighted by the approval of Trastuzumab deruxtecan and Sacituzumab govitecan. These biopharmaceutical approaches have spurred interest in bioconjugation strategies with high and defined degrees of drug-to-antibody ratio (DAR), in particular on native antibodies. Here, a glycoengineering methodology was developed to generate antibody drug conjugates with DAR of up to eight, by combining highly selective enzymatic galactosylation and oxidation with biorthogonal tandem Knoevenagel-Michael addition chemistry. This four-step approach offers a selective route to conjugates from native antibodies with high drug loading, and thus illustrates how biocatalysis can be used for the generation of biopharmaceuticals using mild reaction conditions.
Assuntos
Galactose OxidaseRESUMO
The generation of immobilised oxidase biocatalysts allowing multifunctional oxidation of valuable chemicals using molecular oxygen is described. Engineered galactose oxidase (GOase) variants M1 and M3-5, an engineered choline oxidase (AcCO6) and monoamine oxidase (MAO-N D9) displayed long-term stability and reusability over several weeks when covalently attached on a solid support, outperforming their free counterparts in terms of stability (more than 20 fold), resistance to heat at 60 °C, and tolerance to neat organic solvents such as hexane and toluene. These robust heterogenous oxidation catalysts can be recovered after each reaction and be reused multiple times for the oxidation of different substrates.
RESUMO
Hydride transfer is widespread in nature and has an essential role in applied research. However, the mechanisms of how this transformation occurs in living organisms remain a matter of vigorous debate. Here, we examined dihydrofolate reductase (DHFR), an enzyme that catalyzes hydride from C4' of NADPH to C6 of 7,8-dihydrofolate (H2F). Despite many investigations of the mechanism of this reaction, the contribution of polarization of the π-bond of H2F in driving hydride transfer remains unclear. H2F was stereospecifically labeled with deuterium ß to the reacting center, and ß-deuterium kinetic isotope effects were measured. Our experimental results combined with analysis derived from QM/MM simulations reveal that hydride transfer is triggered by polarization at the C6 of H2F. The σ Cß-H bonds contribute to the buildup of the cationic character during the chemical transformation, and hyperconjugation influences the formation of the transition state. Our findings provide key insights into the hydride transfer mechanism of the DHFR-catalyzed reaction, which is a target for antiproliferative drugs and a paradigmatic model in mechanistic enzymology.