Deracemization of a-Amino Acids

For several reasons a-amino acids are ideal substrates for deracemization methods. They racemize easily by base catalysis under a number of conditions and they are racemized in Nature by the intervention of specific amino acid racemases. They are also recognized as substrates by oxidative enzymes to give the corresponding oxo-acids, in turn substrates for amino transferases and amino acid dehydrogenases. Several industrial preparations of L- and D-amino acids are based on processes of deracemization [26] or of separate two-steps resolution-racemization [27]. 

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Figure 5.4 Deracemization of a-amino acids using D-amino acid oxidase in combination with sodium borohydride.

The method is similar to the deracemization of a-amino acids (see Section... 

In a conceptually related method, the oxidation of the L-lactate is catalyzed by the commercially available L-specific lactate oxidase from Aerococcus viridans and the reduction of pyruvate to rac-lactate is performed with NaBH4 in the same solution. In repeating cycles D-lactate 5 is obtained as the sole product and in an excellent e.e. [16] (Scheme 13.6). This concept has recently been applied to the deracemization of a-amino acids [17]. 

Deracemization of a-Amino Acids via Enzyme-catalyzed DKR Coupled with In Situ Racemization... 

Amino Acid Oxidase and Chemical In Situ Reduction of the Initially Formed Imino Compound A simple and interesting procedure for the deracemization of a-amino acids was introduced by Soda [48], who combined the oxidation of the D-enantiomer of D,L-proline to dehydro-prohne with a chemical reduction of the imine 21 in on -pot, thereby restoring the racemic mixture. If the reaction in the first step is completely enantioselective, the e.e. of the amino acid after one cycle is 50%. Repeating the reaction in successive cycles raises the e.e. close to 100% (Scheme 13.19). 

A common method for the deracemization of a-amino acids has been to employ amino acid oxidases along with a non-selective reduction of the intermediate imine by hydride-reducing agents (e.g., sodium boro hydride or sodium cyanoborohydride) or amine boranes [99, 100]. 

The coupling of these two enzymatic systems could find many more applications due to the avaUabihty of amino acid dehydrogenases of broader specificity [31]. A series of amino acid dehydrogenases with D-specificity for the preparation of D-amino acids has been applied to the reductive amination of a-keto acids [32]. However, the deracemization of rac-amino acids exploiting this type of enzyme requires an amino acid oxidase with L-specificily, which is a rare enzymatic activity. As an alternative the a-oxo acid, usually available through difficult synthetic procedures, can be used directly. 

The procedure reported in Scheme 13.11 describes deracemization of an amino acid involving oxidation with an L-specific enzyme and transamination with a D-amino transferase using D-aspartate 10, which is generated from L-aspartate 11 by aspartate racemase, as the amino donor. The oxidative enzyme is defined as an L-amino acid deaminase, a flavoprotein from Proteus myxofadens [34]. The transamination reaction is shifted towards the product since the oxalacetate 12 formed decarboxylates spontaneously to give pyruvate and carbon dioxide. 

Scheme 11.13 Deracemization of racemic amino acids by cascade reactions, including DAAOs-catalyzed reactions and reductive amination reactions catalyzed either by amino acid dehydrogenases (a) or transaminases (b).

Duhamel L, Plaquevent JC. Deracemization by enantioselective protonation. A new method for the enantiomeric enrichment of a-amino acids. J. Am. Ghent. Soc. 1978 100 7415-7416. 

Recently Turner and coworkers have sought to extend the deracemization method beyond a-amino acids to encompass chiral amines. Chiral amines are increasingly important building blocks for pharmaceutical compounds that are either in clinical development or currently licensed for use as drugs (Figure 5.7). At the outset of this work, it was known that type II monoamine oxidases were able to catalyze the oxidation of simple amines to imines in an analogous fashion to amino acid oxidases. However, monoamine oxidases generally possess narrow substrate specificity and moreover have been only documented to catalyze the oxidation of simple, nonchiral... 

An elegant four-enzyme cascade process was described by Nakajima et al. [28] for the deracemization of an a-amino acid (Scheme 6.13). It involved amine oxidase-catalyzed, (i )-selective oxidation of the amino acid to afford the ammonium salt of the a-keto acid and the unreacted (S)-enantiomer of the substrate. The keto acid then undergoes reductive amination, catalyzed by leucine dehydrogenase, to afford the (S)-amino acid. NADH cofactor regeneration is achieved with formate/FDH. The overall process affords the (S)-enantiomer in 95% yield and 99% e.e. from racemic starting material, formate and molecular oxygen, and the help of three enzymes in concert. A fourth enzyme, catalase, is added to decompose the hydrogen peroxide formed in the first step which otherwise would have a detrimental effect on the enzymes. 

In this chapter deracemization methods for the preparation of multifunctional compounds (a- and P-hydroxy acids, a-hydroxynitriles, and a-amino acids) are discussed (Scheme 13.1). 

A combination of D-amino acid oxidase and L-amino transferase is an example of a deracemization by stereoinversion. The product is an L-amino acid. The reaction catalyzed by amino transferase has an equilibrium constant close to unity, a very unpractical situation leading to uncomplete transformation and to the production of almost inseparable mixtures of amino acids (at least two, the amino acid product and the amino add used as an amino donor). For preparative purposes it is therefore mandatory to shift the equihbrium to the product side. A recent example of a deracemization procedure based on this coupled enzymatic system is the preparation of L-2-naphthyl-alanine 6 as illustrated in Scheme 13.9 [28]. The reaction occurs in one pot with initial oxidation of the D-amino acid catalyzed by D-amino acid oxidase from Rhodotonda gracilis. The hydrogen peroxide that is formed in stoichiometric amounts is decomposed by catalase. The a-keto add is the substrate for L-aspartate amino transferase (L-Asp amino transferase), which is able to use L-cysteine sulfinic acid 7 as an amino donor. 

In a related approach, D,L-methionine can be efSdently deracemized to obtain the L-enantiomer using a multienzyme system consisting of D-amino acid oxidase, catalase, leucine dehydrogenase, and formate dehydrogenase. The a-keto acid 8 produced from the oxidation of the D-form is transformed into L-methionine 9 in the presence of ammonia, leucine dehydrogenase, and a stoichiometric amount of NADH. The NAD thus formed is recycled to NADH with ammonium formate and formate dehydrogenase [30] (Scheme 13.10). 

Hydantoinase-Carbamoylase System for t-Amino Acid Synthesis Despite a number of reports of strains with L-selechve hydantoin-hydrolyzing enzymes [38] the commercial application of the hydantoinase process is stiU restricted to the production of D-amino acids. Processes for the production of L-amino acids are Umited by low space-time yields and high biocatalyst costs. Recently, a new generation of an L-hydantoinase process was developed based on a tailor-made recombinant whole cell biocatalyst. Further reduction of biocatalyst cost by use of recombinant Escherichia coli cells overexpressing hydantoinase, carbamoylase, and hydantoin racemase from Arthrohacter sp. DSM 9771 were achieved. To improve the hydan-toin-converting pathway, the level of expression of the different genes was balanced on the basis of their specific activities. The system has been appUed to the preparation of L-methionine the space-time yield is however still Umited [39]. Improvements in the deracemization process from rac-5-substituted hydantoins to L-amino acids still requires a more selective L-hydantoinase. 

The approach can be coupled with other methods to prepare amino acids, such as to access [3-substituted a-amino acids. The methodology gives a way to prepare all four possible isomers of (3-aryl a-amino acids by a combination of asymmetric hydrogenation and the use of the deracem-ization process to invert the a-center (Scheme 9.36)." "°... 

Scheme 4.37 Deracemization of racemic a-amino acids by combining an enantioselective amino acid oxidase with a non-selective chemical reducing agent.

Other aldehydes have been found to efficiently promote the epimerization of the a-carbon. In 2008, Beller et al. [46] described an efficient deracemization of a-aminoesters, where different aldehydes were screened to evaluate their respective performance. In this study, 3,5-dinitrosalicylaldehyde proved to be particularly efficient for the task, permitting to synthesize several amino acids in high yield and often high enantiomeric excess (Table 8.3). 

Servi, S., Tessaro, D., and Pedrocchi-Fantoni, G. (2008) Chemo-enzymatic deracemization methods for the preparation of enantiopure nonnatural a-amino acids. Coord. Chem. 

Deracemization of p- and y-substituted a-amino acid derivatives by complementary AAOs and l-AADs in combination with different reducing reagents. 

Multienzyme network for the deracemization of 2-amino-3-(7-methyi-1-H-indazol-5-yl)propanoic acid by the combination of a l-AAD with an co-TA. GDH, glucose dehydrogenase LDH, lactate dehydrogenase. 

Flafner et al first reported an oxidase-catalyzed deracemization method using amino acids as the substrate and pkDAAOx or LAAOx from Crotalus adamanteus together with sodium borohydride as the chemical reductant in 1971 [42]. A procedure for the successful deracemization of amino acids was previously reported by Soda et al. [43]. They focused on proline and pipecolic acid as substrates for the production of L-enantiomer by deracemization because these substrates formed stable imines rather than unfavorable keto acids in water by DAAOx. However, the enzyme was denatured by the chemical reaction with sodium borohydride. Turner et al developed an effective production method for (R)- or (S)-amino acids and (S)-amines by a deracemization method using milder chemical reducing reagents such as sodium cyanoborohydride and artificial transfer hydrogenase [44,45]. 

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