Transaminases asymmetric synthesis

Stewart, J.D. (2001) Dehydrogenases and transaminases in asymmetric synthesis. Current Opinion in Chemical Biology, 5, 120-129. 

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. 

Researchers at Celgene developed both (R) and (S) selective transaminases that were active on a range of aliphatic and aromatic ketones and amines [25, 55 57]. Two approaches were employed based upon kinetic resolution, which has been discussed above, and asymmetric synthesis. The asymmetric synthesis approach starts with a... 

Stewart, D. (2001) Dehydrogenases and Transaminases in Asymmetric Synthesis, Curr. Opin. Chem. Biol. 5,120-129. 

On the other hand, a-transaminases have been used extensively in the production of amino acids through kinetic resolution and asymmetric synthesis. While many studies rely on the use of an excess of cosubstrate to drive the reaction to completion, some multienzymatic approaches have been developed as well. As an example, aspartate has been used as an amino donor in a multienzymatic synthesis of L-2-aminobutyrate from L-threonine (Scheme 4.8). ° The rather complex multistep sequence started with the in situ formation of 2-ketobutyrate from L-threonine catalysed by threonine deaminase (ThrDA) from E. coli. A tyrosine transaminase (lyrAT) from E. coli converted 2-ketobutyrate and L-aspartie acid to L-2-aminobutyrate and oxaloacetate, which spontaneously decarboiq lated to give pyruvate. Since the... 

Tufvesson, P., lima-Ramos, J., Jensen, J.S., Al-Haque, N., Neto, W., and Woodley, J.M. (2011) Process considerations for the asymmetric synthesis of chiral amines using transaminases. Biotecknol. Bioeng., 108,1479-1493. 

Finally, great effort has been recently devoted to the exploitation of the so-called co-transaminases (co-TAs) for the preparation of optically pure amines [31]. These biocatalysts are generally pyridoxal-5 -phosphate (PLP)-dependent enzymes and are capable of performing reductive amination readions without using either an a-amino add as amine donor or an a-keto add as amino acceptor. Therefore, they find several applications in the asymmetric synthesis of nonracemic aprimary amines, and enzymes with different substrate spedfidty are currently available also from commercial sources. 

Scheme 11.14 Application ofa-transaminases to the asymmetric synthesis of o-amino acids (a) and the herbicide L-phosphothricin (b).

Hohne, M., Kuhl, S., Karen, R., and Bornscheuer, U.T. (2008) Efficient asymmetric synthesis of chiral amines by combining transaminase and pyruvate decarboxylase. ChemBioChem,... 

D. Koszelewski, I. Lavandera, D. Clay, D. Rozzell, W. Kroutil, Asymmetric synthesis of optically pure pharmacologically relevant amines employing omega-transaminases, Adv. Synth. Catal. 350 (17) (2008) 2761-2766. 

Transaminases are important enzymes in the synthesis of chiral amines, amino acids, and amino alcohols, hi this chapter the properties of transaminases, the reaction mechanisms, and their selectivity and substrate specificity are presented. The synthesis of chiral building blocks for pharmaceutically relevant substances and fine chemicals with transaminases as biocatalysts is discussed. Enzymatic asymmetric synthesis and dynamic resolution are discussed using transaminases. Protein engineering by directed evolution as well as rational design of transaminases under process condition is presented to develop efficient bioprocesses. 

Transaminases can either be uhlized in kinetic resolution or as)unmetric synthesis (Scheme 29.3). Asymmetric synthesis, starting with a prochiral ketone substrate, can theoretically lead to 100% conversion and is usually the preferred route to chiral products (Scheme 29.3a). Furthermore high enantiomeric purity is not dependent on conversion rates, whereas a kinetic resolution (Scheme 29.3b) needs 50% conversion for a high enantiomeric excess (ee). But kinetic resolution is thermodynamically favored, if pyruvate is the amino acceptor, compared to as5munetric synthesis where the equilibrium lies on the substrate side [5,34]. To achieve 100% conversion, dynamic kinetic resolution serves as an alternahve with spontaneous deracemization or the initiation of a suitable racemate for enantiomerically pure substrates (Scheme 29.3c). Deracemization in a one-pot two-step reaction with an (S)-and (R)-selective transaminase, respectively, is a method of choice, but unfortunately two enantiocomplemen-tary enzymes are needed (Scheme 29.3d) [35]. Therefore deracemization with a dehydrogenase in the kinetic resolution step and a transaminase in the following step... 

General synthesis strategies for transaminase-catalyzed reactions. (a) Asymmetric synthesis with transaminase, (b) Kinetic resolution with transaminase, (c) Dynamic kinetic resolution with transaminase, (d) One-pot two-step deracemization with transaminase. 

A drawback of using lactate dehydrogenase as a biocatalyst to remove pyruvate from the reaction equilibrium is the need for the NADH cofactor. Another possibility to eliminate the coproduct is the application of a p5uruvate decarboxylase (Scheme 29.6b). A cofactor is not required, and the resulting products of pyruvate decarboxylation, acetaldehyde, and CO are highly volatile, shifting the equilibrium toward the product [68]. Several pyruvate decarboxylases from yeast and bacteria are commercially available and are active at the same pH value as the transaminase required for the asymmetric synthesis of chiral amines. 

Studies of transaminases in kinetic resolution elucidated several benefits. Compared to asymmetric synthesis the equilibrium favors product formation if pyruvate is used as an amino acceptor. To get enantiopure amines, kinetic resolution is an acceptable choice with yields of 50%. Various (R)- and (S)-selective transaminases are well established nowadays, leading to enantiopure (S)- and (R)-amines, with high ee. Unfortunately product inhibition is one major disadvantage of kinetic resolution. If a critical concentration of product is achieved, the maximum conversion is prevented. Based on kinetic modeling in a previous study from Shin and Kim, the inhibitory effects were based on the strong binding of the product to the PLP cofactor. Consequently the binding of the amino acceptor is hindered, and conversion of the substrate is not possible [72]. 

This makes the -transaminase of O. anthropi an excellent biocatalyst for kinetic resolution of several chiral amines and a promising candidate for asymmetric synthesis. 

The asymmetric synthesis via a-transaminases was described for L-phenylalanine and L-homophenylalanine in several reports and reviews [17,62,86]. Herein the focus is on ffl-transaminases that catalyzed the asymmetric synthesis of optically pure nonproteinogenic amino acids as building blocks for peptidomimetic and other pharmaceutical compounds. The overall advantage of -transaminase-catalyzed reactions is the ability to use achiral amino donors like benzylamine, which thermodynamically favors the equilibriinn toward the product side. 

The asymmetric synthesis of o-amino acids is carried out by an (R)-selective co-transaminase [87]. Several achiral ketones were examined, leading to D-configured amino acids like o-homoalanine, o-serine, o-fluoroalanine, D-alanine, and D-norvaline. A reaction yield of >99% and enanfiopurify of >99.7% were achieved, excepf for D-norvaline. Remarkably, no producf inhibifion by acetophenone was observed at... 

To change the enanhopreference of transaminases, Hohne et al. searched in silico databases and took a closer look at o-selechve and branched-chain transaminases [26]. After cloning the synthehc genes the new enzymes were investigated for selective activity and desired enanhopreference. The ee of the newly identified (R)-selective transaminase was >99.6%, and its achvity was found to be in the same range as the known (S)-selechve hansaminase, which makes it suitable for the asymmetric synthesis of (R)-amines. 

For the in vitro screening of enzyme pairs and enzymatic pathways, immobilized enzyme microreactor systems (lEMR) were established. This includes the reversible immobilization of His -tagged enzymes via nickel-nitriloacetic acid (Ni-NTA) linkage on the surface-derivatized silica. First of all the kinetic parameters for a new enzymatic pathway have to be defined via a transketolase and a w-transaminase in a continuous flow system for a two-step asymmetric synthesis of chiral amino alcohols [147]. The apparenf value is found to be flow rate dependent with different flow rates ranging from 2 to 30 j1/min. The turnover rate was also found to be lower than that in solu-... 

Recently immobilized transaminases in neat organic solvents have been examined for the asymmetric synthesis of amines [153]. The immobilization of the co-transaminase with Sepabeads was carried out by incubation of the transaminase with the beads in buffer for 48 h. By means of the enzyme achvity, the screening of several organic solvents was carried out among which isopropyl acetate was the most suitable solvent. The immobilized enzyme showed high activity and stability under the chosen reachon conditions, as well as good yields of various tested amines. 

Transaminases are most powerful tools for the synthesis of chiral amines, amino acids, and amino alcohols, hi this chapter several approaches for tiie preparation of fine chemicals or building blocks for pharmaceuticals were discussed, like asymmetric synthesis or kinetic resolution. The main limitations of transaminase-catalyzed reactions are the need to shift the equihbrium to the product side and substrate and product inhibition. Some solutions to overcome such inhibition were presented here for example, multienzyme cascades or biphasic extraction of the product. Protein engineering by directed evolution or rational enzyme design is a promising option to find transaminases with different substrate specificities and enantiopreferences. This is becoming more and more important for the pharmaceutical industry. Furthermore, it is a way to alter enzyme properties known so far, like thermostability and solvent and pH stability. Protein engineering has been assisted by the recently solved structures of certain transaminases. 

Dynamic kinetic resolution (DKR) is a method that allows for conversion of the racemic mixture into the desired enantiomer with up to 100% of the theoretical yield. " DKR is a powerful approach to asymmetric synthesis and can be achieved by the application of transition-metal catalysts, Lewis acids, organocatalysts, or enzymes (e.g., hydrolases, dehydrogenases, haloalcohol dehalogenases, and transaminases). 

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