Transaminases enantioselectivity

A more recent study focused on the directed evolution of the co-transaminase from Vibrio fiuvialis JS17, specifically with the aim to eliminate product inhibition by aliphatic ketones while maintaining high enantioselectivity. This was achieved by screening 85 000 clones produced by epPCR [72]. 

Hwang, B.-Y. and Kim, B.-G. (2004) High-throughput screening method for the identification of active and enantioselective m-transaminases. Enzyme and Microbial Technology, 34, 429-436. 

Another class of enzymes that can be used for the enantioselective synthesis of amines and amino acids is the aminotransferases or transaminases (TAs) [29]. As shown in Scheme 6.15, they can be employed in a kinetic resolution or an asymmetric synthesis mode. 

Another case of the use of a single round of mutagenesis to improve the enantioselectivity of an enzyme (which therefore is not an evolutionary process) concerns the enantioselectivity of a transaminase. Transaminases are enzymes which catalyze the conversion of ketones to the corresponding primary amines, the ee values often being >95 % [85]. However, in one case an ee of only 65 % was observed [86], A single library of variants was produced which contained an enzyme showing 98 % ee. Unfortunately... 

A particular example from our laboratory is observed in compound 5, in which a basic amino group held rigidly on a mimic of the coenzyme pyridoxamine phosphate is able to convert ketoacids to amino acids with high enantioselectivity (15) (Fig. 3). This reaction is modeled closely to the way in which transaminase enzymes achieve the same goal. 

Fig. 11. Enantioselective transaminase-catalysed formation of a chiral amine...

Celgene developed transaminase technology for the enantioselective conversion of chiral amines to ketones [25]. Low molecular weight aldehydes, such as propional dehyde, were used as the amine group acceptor. This process has been used on a... 

Hbhne et al. reported a substrate protection strategy that enhanced both the rate and the enantioselectivity of transaminase catalyzed kinetic resolution reactions [32]. The co transaminase catalyzed resolution of the pharmaceutically important syn thons 3 amino pyrrolidine 53 and 3 aminopiperidine 54 was imp roved by the addition of protecting groups to the substrate amines. Reaction rates were improved by up to 50 fold, and product ee was improved from 86 to 99% (Figure 14.23). 

The synthesis of chiral a-amino acids starting from a-keto acids by means of a transamination has been reported by NSC Technologies [26, 27]. In this process, which can be used for the preparation of l- as well as D-amino acids, an amino group is transferred from an inexpensive amino donor, e.g., L-glutamic acid, l-22, or L-aspartic acid, in the presence of a transaminase (= aminotransferase). This reaction requires a cofactor, most commonly pyridoxal phosphate, which is bound to the transaminase. The substrate specificity is broad, allowing the conversion of numerous keto acid substrates under formation of the L-amino acid products with high enantioselectivities [28]. 

In a selection strategy based on the substrate 1-phenyl-n-propylamine (PPA) as the sole source of nitrogen in a chemostat, a recombinant Pseudomonas putida strain carrying the R-transaminase gene, a single amino acid change, Y112F, presumably at or near the active site, improved enantioselectivity of the reaction of racemic... 

A two-step enantioselective synthesis of 2-amino-l,3.4-butanetriol 12 was performed in continuous mode using two serial capillary microreactors with HiSg-tagged transketolase (TK) /oo-transaminase (TAM) bound to the wall via immobihzed Ni-nitrilotriacetic add (Ni-NTA) complex [78]. The TK-catalyzed conversion of hydroxypyruvate 8 and glycolaldehyde 9 to L-erythrulose 10 followed by the TAM-catalyzed amination resulted in the formation of the product This work demonstrated the implementation of a dual enzyme microreactor system for the evaluation of a de novo pathway for an enzyme-catalyzed synthesis. 

Another possible cascade reaction would be the combination of nitrilases and transaminases, in principle again exploiting enantioselective synergy. Similarly the combined use of nitrilases with acylases or Hpases is conceivable, when working with N-acylated substrates. However, the size of the acyl group should be compatible with the nitrilase active site. 

Another relevant example is provided by the enzymatic s)mthesis of the antidiabetic, sitagliptin, which was codeveloped by Merck and Codexis workers [67] to replace a rhodium-catalyzed, high-pressure, as5mimetric hydrogenation of an enamine. If involves an overall enantioselective reductive amination of a ketone using an (R)-transaminase-catalyzed reaction with isopropylamine (Figure 1.5). The starting point was an (R)-selective transaminase, which showed no activity with the ketone substrate. In silico studies were employed to identify whaf was needed to be able to fif fhe... 

The most frequently described transaminases show high enantioselectivity for the chiral carbon atom with the carboxylic group at the a-position or the chiral center at the p-position. To determine the mechanism toward enantioselectivity of transaminases, a structural analysis is necessary. The crucial factor is the architecture of tiie active... 

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