The use of transaminases to access pharmaceutically relevant chiral amines is an attractive alternative to transition-metal-catalysed asymmetric chemical synthesis. However, one major challenge is their limited substrate scope. Here we report the creation of highly active and stereoselective transaminases starting from fold class I. The transaminases were developed by extensive protein engineering followed by optimization of the identified motif. The resulting enzymes exhibited up to 8,900-fold higher activity than the starting scaffold and are highly stereoselective (up to >99.9% enantiomeric excess) in the asymmetric synthesis of a set of chiral amines bearing bulky substituents. These enzymes should therefore be suitable for use in the synthesis of a wide array of potential intermediates for pharmaceuticals. We also show that the motif can be engineered into other protein scaffolds with sequence identities as low as 70%, and as such should have a broad impact in the field of biocatalytic synthesis and enzyme engineering.
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...
The evolution of the synthesis of oseltamivir phosphate (Tamiflu ® ), used for the oral treatment and prevention of influenza virus infections (viral flu) is described. Oseltamivir phosphate is the ethyl ester prodrug of the corresponding acid, a potent and selective inhibitor of influenza neuraminidase. The discovery chemistry route and scalable routes used for kilo laboratory production as well as the technical access to oseltamivir phosphate from (-)-shikimic acid proceeding via a synthetically well-developed epoxide building block followed by azide transformations are reviewed. Synthesis and process research investigations towards azide-free conversions of the key epoxide building block to oseltamivir phosphate are discussed. The search for new routes to oseltamivir phosphate independent of shikimic acid including Diels-Alder approaches and transformations of aromatic rings employing a desymmetrization concept are presented in view of large-scale production requirements.
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