Amino acid oxidases deracemization

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

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... 

Chemoenzymatic processes involving oxidizing enzymes have been reported particularly for specific chemical syntheses. For example, industrially important amino acids can be deracemized by exploiting the enantioselectivity of amino acid oxidases a commercial process has recently been developed in which efficient... 

Deracemization of amino-acids One-pot h2o NaCNBH3 or NaBE, Amino acid oxidase 2,7,14... 

Deracemization by Stereoinversion via the Two-enzyme System D-Amino Acid Oxidase and L-Amino Transferase... 

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). 

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. 

Deracemization by Stereoinversion via the Three-enzyme System i-Amino Acid Oxidase, D-Amino Transferase and Amino Add Racemase... 

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). 

Thus, using L-amino add oxidase from P. myxcfaciens and various amine-borane complexes or D-amino acid oxidase from porcine kidney and sodium cyanoboro-hydride, the preparation of several natural and non-natural enantiopure D- and L-amino adds was achieved, respectively [51]. In a more recent report, several P- and y-substituted a-amino adds were deracemized using D-amino add oxidase from Trigonopsis variahilis and sodium cyanoborohydride or sodium borohydride [52] (Scheme 13.20). 

Scheme 13.19 Chemoenzymatic deracemization with D-amino acid oxidase and chemical reduction.

Enzymatic oxidations of carbon-nitrogen bonds are as diverse as the substances containing this structural element. Mainly amine and amino acid oxidases are reported for the oxidation of C-N bonds. The steroespecificity of amine-oxidizing enzymes can be exploited to perform resolutions and even deracemizations or stereoinversions (Fig. 16.7-1 A). Analogous to the oxidation of alcohols, primary amines are oxidized to the corresponding imines, which can hydrolyze and react with unreacted amines (Fig. 16.7-1 B). In contrast to ethers, internal C-N bonds are readily oxidized, yielding substituted imines. This can be exploited for the production of substituted pyridines (Fig. 16.7-1 C). Furthermore, pyridines can be oxidized not only to N-oxides but also to a-hydroxylated products (Fig. 16.7-1 D). 

Figure 16.7-8. Enzymatic deracemization of amino acids catalyzed by D-amino acid oxidase (d-AAO). Leucine dehydrogenase (LeuDH) transforms the oxidation product of the undesired amino acid enantiomer in situ into the racemic amino acid. Regeneration of NADH is performed by formate dehydrogenase (FHD).

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

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]. 

S)-Amino-3-[3- 6-(2-methylphenyl) pyridyl]-propionic add 82a was prepared by an enzymatic deracemization process using a combination of two enzymes (P)-amino acid oxidase from Trigonopsis variabilis cloned and expressed in E. coli and an (S)-aminotransferase from Sporosarcina ureae, also cloned and expressed in E. coli [147]. Racemic amino acid 82 was used as a substrate and (S)-aspartate was used as amino donor. An (S)-aminotransferase was also purified from a soil organism identified as Burkholderia sp. and cloned and expressed in E. coli and used in this process [147]. This process was scaled up to 70 L scale. 

Yasukawa, K., Nakano, S., and Asano, Y, "Tailoring D-Amino acid oxidase from the pig kidney to R-stereoselective amine oxidase and its use in the deracemization of -methylbenzylamine." Angew. Chem. Int. Ed., 53, 4428-4431 (2014). 

Alexandre, R R., Pantaleone, D. R, Taylor, P. R, Fotheringham, I. G., Ager, D. J., and Turner, N. J., "Amine-borane effective reducing agents for the deracemization of OL-amino acids using L-amino acid oxidase from Proteus myxofaciens." Tetrahedron Lett., 43,707-710 (2002). 

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 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]. 

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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