Biocatalysis has emerged as a powerful alternative to traditional chemistry, especially for asymmetric synthesis. One key requirement during process development is the discovery of a biocatalyst with an appropriate enantiopreference and enantioselectivity, which can be achieved, for instance, by protein engineering or screening of metagenome libraries. We have developed an in silico strategy for a sequence-based prediction of substrate specificity and enantiopreference. First, we used rational protein design to predict key amino acid substitutions that indicate the desired activity. Then, we searched protein databases for proteins already carrying these mutations instead of constructing the corresponding mutants in the laboratory. This methodology exploits the fact that naturally evolved proteins have undergone selection over millions of years, which has resulted in highly optimized catalysts. Using this in silico approach, we have discovered 17 (R)-selective amine transaminases, which catalyzed the synthesis of several (R)-amines with excellent optical purity up to >99% enantiomeric excess.

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Scheme 1. Asymmetric hydrogenation and addition are the two main synthetic strategies in the chemical preparation of a-chiral primary amines. If chiral auxiliaries are used to generate enantioselectivity, R 3 represents a chiral substituent. Deprotection is an important step in obtaining the desired amine, so the choice of a readily cleavable group, such as diphenylphosphinyl or tert-butylsulfinyl, is recommended. [23] Scheme 2. Summary of possible enzymatic routes to optically active a-chiral amines.Scheme 6. Asymmetric synthesis of (S)-2-amino-1-methoxypropane.

For the biocatalytic preparation of optically active amines, omega-transaminases (omega-TA) are of special interest since they allow the asymmetric synthesis starting from prostereogenic ketones with 100% yield. To facilitate the purification and characterization of novel omega-TA, a fast kinetic assay was developed based on the conversion of the widely used model substrate alpha-methylbenzylamine, which is commonly accepted by most of the known omega-TAs. The product from this reaction, acetophenone, can be detected spectrophotometrically at 245 nm with high sensitivity (epsilon = 12 mM(-1) cm(-1)), since the other reactants show only a low absorbance. Besides the standard substrate pyruvate, all low-absorbing ketones, aldehydes, or keto acids can be used as cosubstrates, and thus the amino acceptor specificity of a given omega-TA can be obtained quickly. Furthermore, the assay allows the fast investigation of enzymatic properties like pH and temperature optimum and stability. This method was used for the characterization of a novel omega-TA cloned from Rhodobacter sphaeroides, and the data obtained were in excellent accordance with a standard capillary electrophoresis assay.

Technological developments enable the discovery of novel enzymes, the advancement of enzyme cascade designs and pathway engineering, moving biocatalysis into an era of technology integration, intelligent manufacturing and enzymatic total synthesis.

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Two is better than one (enzyme). The combination of ω‐transaminase with pyruvate decarboxylase (PDC) allowed for the efficient asymmetric synthesis of chiral amines, because the unfavorable equilibrium could be shifted by removal of pyruvate by using PDC.

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...