Recent investigations on imine reductases (IREDs) have enriched the toolbox of potential catalysts for accessing chiral amines, which are important building blocks for the pharmaceutical industry. Herein, we describe the characterization of 20 new IREDs. A C-terminal domain clustering of the bacterial protein-sequence space was performed to identify the novel IRED candidates. Each of the identified enzymes was characterized against a set of nine cyclic imine model substrates. A refined clustering towards putative active-site residues was performed and was consistent both with our screening and previously reported results. Finally, preparative scale experiments on a 100 mg scale with two purified IREDs, IR_20 from Streptomyces tsukubaensis and IR_23 from Streptomyces vidiochromogenes, were carried out to provide (R)-2-methylpiperidine in 98% ee (71% yield) and (R)-1-methyl-1,2,3,4-tetrahydroisoquinoline in >98% ee (82% yield).
Biocatalysis employing imine reductases is ap romising approach for the one-step generation of chiral amines from ketones. The enzymes reported for this process suffer from low activity and moderate stereoselectivity.W ei dentified as et of enzymest hat facilitate this reaction with high to quantitative conversionsf rom al ibrary of 28 imine reductases. This enabled the conversion of ketones with ammonia, methylamine, or butylamine into the corresponding amines. Most importantly,w e performed preparative (> 100 mg) scale syntheses of amines such as (1S,3R)-N,3-dimethylcyclohexylamine and (R)-N-methyl-2-aminohexane with excellent stereochemical purities (98 % de, 96 % ee)i ng ood yields.Chiral amines are keys tructuralm otifs that are frequently employedf or the preparation of pharmacologically active compounds.[1] From av ariety of asymmetric synthesis methods, transition-metal-catalyzed reductionso fi mine or enamine precursors [2] dominate the spectrum of approaches that have been reported.[3] However,t hese reactions equences usually comprise severals teps, including protection and deprotection of the amine and the necessity to introduce nitrogen activating groups for the reduction (Scheme 1, routes Aand B). [2] Biocatalysis offers an attractive alternative for synthetic chemists, with ac ontinuouslyi ncreasing toolboxo fe nzymes for the preparation of chiral amines. [1b, 4] Amine transaminases [1b, 5] and amine dehydrogenases, [6] for example, have proven to be applicable in the one-step preparation of primary amines,a lso on industrial scale. [5c, 7] Thus, these enzymes can providea na ttractive synthetic shortcut towards chiral amines. Additionally,C odexis lately filed ap atento ne ngineered opine dehydrogenases that catalyze reductive aminations.[8] NADPHdependenti mine reductases (IREDs) also catalyze the equivalent single-step reduction (Scheme 1, route C) but are, unlike amine transaminases anda mine dehydrogenases, not limited to the preparation of primary amines. IREDs are amenable to access primary,secondary,and tertiary amines starting from ketones or the respective imine or iminium ion. Whereas imine reductionsh ave been knownf or some time to occur in several metabolic pathways such as dihydrofolate, opine, opioid, and antibiotic synthesis, [9] it was only over the last five years that NADPH-dependentI REDs were reported in the context of synthetic applications.[10] Immediately after the isolation of the first amino acid sequences, [10] several research groups thoroughly studied IREDs for the reduction of variousc yclic imines and iminium ion substrates, also including artificial metal-dependentI REDs.[11] Very recently,t he group of Müllerr eported the first IRED-catalyzed reductivea mination of ak etone with methylamine in ap roofo fp rinciple study.[11e] With this work, he paved the way for an ew synthetic application of IREDs. Nestl and co-workersd emonstrated that IRED-catalyzedr eductive amination is, in principle, applicable for different amines, which thuso pened the gate for the sy...
NADP(H)-dependent imine reductases (IREDs) are of interest in biocatalytic research due to their ability to generate chiral amines from imine/iminium substrates. In reaction protocols involving IREDs, glucose dehydrogenase (GDH) is generally used to regenerate the expensive cofactor NADPH by oxidation of d-glucose to gluconolactone. We have characterized different IREDs with regard to reduction of a set of bicyclic iminium compounds and have utilized H NMR and GC analyses to determine degree of substrate conversion and product enantiomeric excess (ee). All IREDs reduced the tested iminium compounds to the corresponding chiral amines. Blank experiments without IREDs also showed substrate conversion, however, thus suggesting an iminium reductase activity of GDH. This unexpected observation was confirmed by additional experiments with GDHs of different origin. The reduction of C=N bonds with good levels of conversion (>50 %) and excellent enantioselectivity (up to >99 % ee) by GDH represents a promiscuous catalytic activity of this enzyme.
Stereoselective catalysts for the Pictet-Spengler reactiono ft ryptamines and aldehydesm ay allow as imple and fast approach to chiral 1-substitutedt etrahydro-b-carbolines. Although biocatalystsh ave previously been employed for the Pictet-Spengler reaction,not as ingle one acceptsb enzaldehyde and its substituted derivatives. To addresst his challenge,acombination of substrate walking and transfer of beneficial mutationsb etween different wild-type backbones was used to develop as trictosidine synthase from Rauvolfia serpentina (RsSTR) into as uitable enzymef or the asymmetricP ictet-Spengler condensation of tryptaminea nd benzaldehyde derivatives. The double variant RsSTR V176L/V208A accepted various ortho-, metaand para-substituted benzaldehydes and produced the correspondingc hiral 1-aryl-tetrahydro-b-carbolines with up to 99 %enantiomeric excess. Scheme1.Biocatalytic Pictet-Spenglerr eaction using astrictosidine synthase variant to transformbenzaldehyde. (A) The wild-typeSTR does not transformbenzaldehyde. (B) Substrate scope of RsSTR V176L/V208.
Enzymes are valuable tools to introduce chirality into small molecules. Especially, ketoreductase (KRED)-catalyzed transformations of ketones to yield chiral secondary alcohols have become an established biocatalytic process step in the pharmaceutical and fine chemical industry. Development time, however, remains a critical factor in chemical process development and thus, the competitiveness of a biocatalytic reaction step is often governed by the availability of off-the-shelf enzyme libraries. To expand the biocatalytic toolbox with additional ketoreductases, we established a multi-faceted screening procedure to capture KRED diversity from different sources, such as literature, available genome data, and uncharacterized microbial strains. Overall, we built a library consisting of 51 KRED enzymes, 29 of which have never been described in literature before. Notably, 18 of the newly described enzymes exhibited anti-Prelog preference complementing the majority of ketoreductases which generally follow Prelog's rule. Analysis of the library's catalytic activity toward a chemically diverse ketone substrate set of pharmaceutical interest further highlighted the broad substrate scope and the complementing enantio-preference of the individual KREDs. Using the generated sequence-function data of the included short chain dehydrogenases in a bioinformatic analysis led to the identification of possible sequence determinants of the stereospecificity exhibited by these enzymes.
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