Amidase amino acid synthesis

Scheme 2.11 Synthesis of amino acids from amino nitriles using a nitrile hydratase and amidase.

Recently a number of enzymatic systems have been developed at several chemical companies including Upases (synthesis of enantiotrope alcohols, R-amid, S-amin), nitrilases (R-mandehc acid), amidases (non-proteinogenic L-amino acids), aspartic acid ammonia lyase (L-aspartic add), penicilin acylase (6-Aminopenicilanic acid), acylases (semisynthetic penicillins), etc.( Koeller and Wong, 2001 and references therin). 

DSM developed a slightly different approach towards enantiopure amino acids. Instead of performing the Strecker synthesis with a complete hydrolysis of the nitrile to the acid it is stopped at the amide stage. Then a stereoselective amino acid amidase from Pseudomonas putida is employed for the enantioselective second hydrolysis step [83], yielding enantiopure amino acids [34, 77, 78]. Although the reaction is a kinetic resolution and thus the yields are never higher than 50% this approach is overall more efficient. No acylation step is necessary and the atom efficiency is thus much higher. A drawback is that the racemisation has to be performed via the Schiff s base of the D-amide (Scheme 6.23). 

The hydrolysis of amides is not limited to the industrial synthesis of enantio-pure amino acids. Lonza has developed routes towards (S)-pipecolic acid and (R)- and (S)-piperazine-2-carboxylic acid that are based on amidases [93, 94]. 

Ma, D.Y, Wang, D.X., Pan, J. et al. 2008a. Nitrile biotransformations for the synthesis of highly enantioenriched P-hydroxy and P-amino acid and amide derivatives A general and simple but powerful and efficient benzyl protection strategy to increase enantiose-lectivity of the amidase. Journal of Organic Chemistry, 73 4087 091. 

Although cephalosporin C is divisable into a-aminoadipic acid, cysteine, and valine, the actual mechanism whereby Cephalosporium sp. incorporates the three amino acids into cephalosporin C has not been established, Arnstein and Morris isolated 8 (a-aminoadipyl) cysteinyl valine from mycelia of Penicillium chrysogenum and suggested that the tripeptide is a precursor in all penicillin biosynthesis.. This same tripeptide also appears to be found in the intracellular pool of Cephalosporium sp.- The final postulated step in the biosynthesis of penicillin is an acyl transfer reaction, or the production of 6-aminopeni-cillanic acid if precursor is not added. Cephalosporium sp. apparently do not produce sidechain amidases or acyl transferases, and no 7-ACA has been reported found in the fermentation. Thus, to obtain clinically useful antibiotics, chemical manipulation of cephalosporin C is necessary. Synthesis of many 7-acyl derivatives was possible once a practical cleavage reaction made available large amounts of 7-ACA from cephalosporin C. of these derivatives, sodium cephalothin was the first... 

A classic example of a typical enzymatic resolution on an industrial scale is the acylase-mediated production of L-methionine. This method has also been applied for the production of L-phenylalanine and L-valine. In addition to acylases, amidases, hydantoinases, and /i-lactam hydrolases represent versatile biocatalysts for the production of optically active L-amino acids. A schematic overview of the different type of enzymatic resolutions for the synthesis of L-amino acids is given in Fig. 2. 

It should also be noted that at DSM this amidase process has been extended towards the synthesis of optically active a,a-disubstituted amino acids. For example, the antihypertensive drug L-methyl-dopa, l-10, has been produced successfully (Fig. 8) [14, 15]. 

An example of a very efficient asynunetric transformation is the preparation of (f )-phenylgly-cine amide (8) where the synthesis of the amino acids, by a Strecker reaction, and racemization are aU performed at the same time (Scheme 9.9). This offers a good alternative to the enzymatic resolution of phenylglycine amide with an (S)-specific amidase. ... 

Interesting new enzymes can of course also be isolated from non-microbial sources. A recent example of a useful novel enzyme or plant origin is the peptide amidase from orange flavedo (Fig. 5) discovered by Steinke and Kula [130, 131]. This enzyme, which has an extremely wide substrate range, is useful for C-terminal enzymatic deprotection in peptide synthesis under very mild conditions. Substrates of this peptide amidase are protected and unprotected peptide amides, N-protected amino acid amides. The enzyme is stereoselective with regard to the C-terminal position only L-amino acid amides are accepted as substrates, with the exception of proline. Other interesting enzymes of plant origin are the oxynitrilases (see below). 

Enzymatic Resolution of Racemic a-Methyl Phenylalanine Amides. The chiral amino acids (22) and (23) (Fig. 6A) are intermediates for the synthesis of (33-receptor agonists (30,31). These are available via the enzymatic resolution of racemic a-methyl phenylalanine amide (24) and a-methyl-4-methoxy-phenylalanine amide (25), respectively, by an amidase from Mycobacterium neoaurum ATCC 25795 (32). Wet cells (10% wt/vol) completed the reaction of amide (24) in 75 min with a... 

Desymmetrization of Prochiral Dinitriles. Prochiral a,a-disubstituted malono-nitriles can be hydrolyzed in an asymmetric manner by the aid of Rhodococcus rhodochrous [678] (Scheme 2.106). In accordance with the above-mentioned trend, the dinitrile was nonselectively hydrolyzed by the nitrile hydratase in the cells to give the dicarboxamide. In a second consecutive step, the latter was subsequently transformed by the amidase with high selectivity for the pro-(/ ) amide group to yield the (/ )-amide-acid in 96% e.e. and 92% yield. This pathway was confirmed by the fact that identical results were obtained when the dicarboxamide was used as substrate. The nonracemic amide-acid product thus obtained serves as a starting material for the synthesis of nonnatural a-methyl-a-amino acids [679]. 

The hydrolytic enzymes amidases are useful for the hydrolysis ofN-acylamino acids for the synthesis of amino acids and in the formation of amide bonds in polypeptides and proteins. In fact, this method is the resolution of amino acids. ... 

Amidases are also applied for the chiral resolution of racemic amino acid amides to allow the biocatalytic synthesis of nonnatural i.-amino acids, which are important building blocks for pharmaceuticals. An amidase (EC 3.5.1.4) from Pseudomonas putida has been developed for the kinetic resolution of a wide range of amino acid amides (Schmid et al. 2001). 

Synthesis of (S)- and (/ )-a-amino acids using nitrile hydratase (NHase) from Rhodococcus opacus, a-amino-e-caprolactam racemase (ACL) from Achromobacter obae, and different amidases (amidase 1, o-amino-peptidase from Ochrobactrum anthropi amidase 2, i-amino acid amidase from Brevundimonas diminuta) [111, 112]. / =CH3, CH CH, CH3CH(CH3), CH3CH(CH3)CH2. 

Barbas, C. F., Wong, C.-H. (1987). Papain catalysed peptide synthesis control of amidase activity and the introduction of unusual amino acids. Chem. Commun., Issue 8,533-534. 

In the kinetic resolution of amino acid amides with the use of amidases, such as DAP and DaaA, it is possible to synthesize D-amino acids by kinetic resolution, selectively from racemic acid amides [16]. An Escherichia coli transformant highly expressing DAP catalyzed the synthesis of 2.5M (about 220g/l) D-alanine from 5M racemic alanine amide in a 4.5-h reaction, d-2-Amino butyric acid, D-methionine, D-norvaline, and D-norleucine were S5mthesized in a similar manner. We have been successful in the evolution of DAP [17] and DaaA by mutations [18]. 

Yamaguchi, S., Komeda, H., and Asano, Y., "New enzymatic method of chiral amino add synthesis by dynamic kinetic resolution of amino acid amides use of stereoselective amino acid amidases in the presence of a-amino-e-caprolactam racemase." Appl. Environ. Microbiol, 73,5370-5373 (2007). 

In this chapter we describe the DSM aminoamidase processes in more detail. Three different enzymatic resolution routes have been developed for the preparation of natural and synthetic amino acids using biocatalysts from different origin, i.e.. Pseudomonas pu-tida, Mycobacterium neoaurum, and Ochrobactrum anthropi. Scope and limitations and enzyme characterization of these amidases will be presented together with some specific examples. In addition, the use of some of these amino acids in peptide sjmthesis, catalytic asymmetric synthesis, and further synthetic transformations will be given. 

Figure 34 Synthesis of L-a-alkyl amino acids by amidase.

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