Transaminases kinetic resolution

Yun, H., Cho, B.-K. and Kim, B.-G., Kinetic resolution of (R,S)-jec-butylamine using omega-transaminase from Vibrio fluvialis JS17 under reduced pressure. Biotechnol. Bioene., 2004, 87, 772-778. 

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. 

Recently, a lipase-catalysed kinetic resolution has been developed to produce L-tert-leucine via a lactone of its A-benzoyl derivative. There is also reputed to be another bioroute to L-terMeucine using transaminase technology. 

While kinetic resolution with the help of lipases or esterases has seen the greatest success for the synthesis of enantiomerically pure amines, the same target can be reached by employing co-transaminases (co-TA) to reductively transaminate ketones to either (S)- or (K)-amines, depending on the transaminase. The reaction is shown in Figure 7.22 with acetophenone and (S)-transaminase as an example (Shin, 1998, 1999). 

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). 

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... 

While most methods for the synthesis of enantiomerically pure amines have employed kinetic resolution with the help of lipases or esterases, a method independent of kinetic resolution has been developed using the transamination of ketones catalyzed by co-transaminases ([Pg.880]

Scheme 4.12 co-Transaminase-catalyzed amination of a carbaldehyde in a dynamic kinetic resolution with subsequent lactamization cascade to provide (R)-4-phenyipyrroiidin-2-one. 

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... 

Koszelewski, D., Clay, D., Faber, K., and Kroutil, W. (2009) Synthesis of 4-phenylpyrrolidin-2-one via dynamic kinetic resolution catalyzed by OJ-transaminases. 

Like amine oxidases, one can also combine amino add dehydrogenases with in situ chemical reduction, a transaminase, or an amino add dehydrogenase to effect dynamic kinetic resolutions of amino adds. Details of these more complex processes are desaibed in the appropriate later chapters. 

M.D.Truppo, N.. Turner, J.D. Rozzell, Efficient kinetic resolution ofracemic amines using a transaminase in combination with an amino acid oxidase Chem. Commun. (16) (2009) 2127-2129. 

While first examples regarding the biocatalytic preparation of chiral amines mainly involved kinetic resolution processes using hydrolases [15], in the last years, the identification of novel biocatalysts including amine oxidases (AOs), amino acid oxidases (AAOs), lyases, or ra-transaminases (co-TAs) has provided new catalytic tools for the production of chiral amines with high stereoselectivity [16-18]. 

Another example, from the Pfizer portfolio, of successful incorporation of biocatalysis to make functional group interconversions (FGIs) more efficient in API synthesis is in a program for a smoothened (SMO) receptor inhibitor [17], Introduction of a transaminase-catalyzed stereoselective reductive amination of a 4-piperidone 4 with concurrent dynamic kinetic resolution (DKR) gave amine 5 (Scheme 7.2), resulting in the highly efficient incorporation of two chiral centers in a single step. 

Chiral amines have been attracting attention as an important composition, particularly for pharmaceutical products. The organic synthetic methods of optically active amine compounds have been developed through the traditional resolution of racemic amines with the formation of diastereomer salts using an optically active mandelic acid or tartaric acid. Enzymatic synthesis has mainly used lipase and S- or R-stereoselective amine transaminase (AT) [29-31] (Figure 19.7). Turner et al. successfully synthesized chiral (R)- and (S)-amines by kinetic resolution using a combination of stereoselective AT and d- or L-amino acid oxidase (AAOx) [32] (Figure 19.7). However, the theoretical yield of the products has been limited to 50% in the kinetic resolution. 

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. 

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]. 

In addition the transaminase from O. anthropi showed no substrate inhibition up to 500 mM of (S)-a-methylbenzylamine in any cases. For the kinetic resolution with 500 mM (S)-a-methylbenzylamine, 300mM pyruvate, and 75U/ml of O. anthropi transaminase, reaction yield of 95.3% and ee values of >99% were obtained after 3h compared to the transaminase of P. denitrificans vith no detectable conversion and the ee values of <22% obtained after 10 h. 

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

Another approach has recently been described by Cuetos et al. for the dynamic kinetic resolution of a-substituted p-amino esters [77]. Several ra-transaminases and acyclic alkyl-p-keto esters were tested, leading to high conversion rates and high ee and de values. With this result in mind it seems feasible that novel transaminases will be designed to create enantiomeric pure molecules under mild and economically feasible conditions. 

The best conversion 3deld in the kinetic resolution step, up to 55%, and ee values of >99% were achieved with 50 mM roc-mexiletine. After defining the best reaction conditions, the two reachon steps were combined by adding the -transaminase with opposite enanhopreference. Unfortunately it was shown that the -transaminase used in the first step also catalyzed the reduchve aminahon reaction. To overcome this problem, prior to the addihon of the transaminase with the opposite enanhopreference, the hansaminase used in the first step was kept at 75 °C for 30 min. The combination of the -hansaminase horn C. violaceum and the transaminase ATA-117 achieved excellent conversion 3deld and ee values of >99% for the kinetic resoluhon and the reduchve aminahon step, respechvely. 

The advantage of the kinetic resolution of p-amino acids is the shift of the equilibrium toward the product side, due to the spontaneous decarboxylation of tire p-keto acid by-product. For the synthesis of D-amino-N-butyric acid with an co-transaminase from Alcaligenes denitrificans and pyruvate as amino acceptor, conversion yields of 53% and >99% ee were obtained [52]. The kinetic resolution of racemic p-phenyla-lanine catalyzed by the co-transanrinase from Burkholderia phytoflrmans was examined recently [101]. 

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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