Transaminases chiral amines

Deamination, Transamination. Two kiads of deamination that have been observed are hydrolytic, eg, the conversion of L-tyrosiae to 4-hydroxyphenyUactic acid ia 90% yield (86), and oxidative (12,87,88), eg, isoguanine to xanthine and formycia A to formycia B. Transaminases have been developed as biocatalysts for the synthetic production of chiral amines and the resolution of racemic amines (89). The reaction possibiUties are illustrated for the stereospecific synthesis of (T)-a-phenylethylamine [98-84-0] (ee of 99%) (40) from (41) by an (5)-aminotransferase or by the resolution of the racemic amine (42) by an (R)-aminotransferase. 

Shin, J.S. and Kim, B.G., Comparison of the ra-transaminases from different microorganisms and application to production of chiral amines. Biosci. Biotechnol. Biochem., 2001, 65, 1782-1788. 

A transaminase patented by Celgene Corporation (Warren, NJ), called an co-aminotransferase [(co-AT)E.C. 2.6.1.18] does not require an a-amino acid as amino donor instead it requires a primary amine and hence has the ability to produce chiral amines.125 126 A similar co-AT from Vibrio fluvialis has been described for the production of chiral amines along with chiral alcohols when coupled with AdH or chiral amino acids when coupled with an a-amino acid aminotransferase.127130 Another co-AT, ornithine (lysine) aminotransferase (E.C. 2.6.1.68), has been described for the preparation of a chiral pharmaceutical intermediate used in the synthesis of Omapatrilat, a vasopep-tidase inhibitor developed by Bristol-Myers Squibb, as well as the UAA A1 -piperidinc-6-carboxylic acid.131-132... 

The aminotransferase, or transaminase class of enzymes, are ubiquitous, PLP-requiring enzymes that have been used extensively to prepare natural L-amino acids [84,85]. They catalyze the general reaction shown in Scheme 15, where an amino group from one L-amino acid is transferred to an a-keto acid to produce a new L-amino acid and the respective a-keto acid. Those enzymes most commonly used have been cloned, overexpressed, and generally used as whole cell or immobilized preparations. These include the following branched chain aminotransferase (SCAT) (EC 2.6.1.42), aspartate aminotransferase (AAT) (EC 2.6.1.1), and tyrosine aminotransferase (TAT) (EC 2.6.1.5). A transaminase patented by Celgene Corporation (Warren. NJ), called an co-aminotransferase, does not require an a-amino acid as amino donor and hence is used to produce chiral amines [86,87]. Another useful transaminase, n-amino acid transaminase (DAT) (EC 2.6.1.21), has been the subject of much study [37,88,89]. This enzyme catalyzes the reaction using a n-amino acid donor, either alanine or aspartate (Scheme 16). 

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... 

Figure 14.22 Resolution of racemic chiral amines using a transaminase from 6. megaterium.

Figure 14,45 Synthesis of a range of chiral amines using transaminases.

Outline the problems associated with transaminase catalyzed amination of ketones to produce chiral amines, particularly with respect to product inhibition and equilibrium. Suggest two different methods that can be used to overcome these problems. 

Depending on the substrate preference of the employed transaminase, the following couples of sacrificial amine donor/keto acceptors were used, which are often derived from the a-aminoacid pool (such as alanine/pyruvate, phenylalanine/ phenylpyruvate, glutamic acid/a-ketoglutarate, aspartic acid/a-ketosuccinate) or constitute simple amines/ketones, such as 2-propylamine/acetone and 2-butyl-amine/2-butanone. It should be kept in mind that the absolute configuration of a chiral amine-donor has to match the stereospecificity of the co-TA in order to be accepted. 

Figure 15.6 Chiral amines accessible from ketones using o-transaminases and amine donors (typically alanine, isopropyl amine, a-methylbenzylamine).

A typical process flow diagram for the production of chiral amines using aminotransferases is shown in Figure 7.8. The buffering agent, pyridoxal 5-phosphate, amine donor, enzyme, and substrate ketone in aqueous solution are mixed in a biotransformation vessel. The desired reaction conditions such as temperature, pH, amine donor, and acceptor concentrations are maintained during transamination reaction. The transamination reaction time depends on the rate at which transaminase is catalyzing the reaction, while the extent of conversion depends on... 

In order to scale up the transaminase-catalyzed chiral amine production process, process economics is an important aspect with scientific and technical aspects involved. In a given transaminase process, the cost of a product or production cost can be expressed in a simplified form as follows [1]. 

If the transaminase is used in the form of whole cells instead of purified enzyme, the cost of production of chiral amines decreases, since enzyme purification adds... 

When the transaminase-catalyzed chiral amine synthesis is identified on a small scale, there are a number of factors that are required to be addressed in order to... 

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. 

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

Another important field of application of NAD(P)H-dependent dehydrogenases is the co-transaminase (co-TA)-catalyzed processes for the preparation of chiral amines. 

The enzyme class which has received the most increase in attention in industrial and synthetic applications since 2000 is (o-transaminases, in which a suitable inexpensive amino-donor 16 is used to convert a C=0 group of a prochiral substrate 15 into chiral amine 17. In order to shift the equilibrium to the desired amine product, the keto by-product 18 is usually converted in a second irreversible enzymatic step into an iimocent side product 19 (Scheme 15.3). 

Cofactor regeneration is not only a task when using oxidoredurtases but also for some representatives from other enzyme classes, for example, transferases and lyases. Transaminases require pyridoxal phosphate and transform a ketone into the chiral amine on the expense of a donor amine that is the cosubstrate needed in stoichiometric amount [17]. A representative example for pyridoxal phosphate regeneration is shown in Scheme 2.5, exemplified for the use of isopropylamine as an amine donor. This reagent is especially useful for two reasons. First, it is a small (and thus atom economical), cheap, and readily available amine. 

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]. 

SYNTHESIS OF CHIRAL AMINES USING o-TRANSAMINASES 2.2.1 ct)-Transaminases Definition and General Facts... 

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