Amidase from Pseudomonas

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). 

In addition, at Ube an analogous process for the enantioselective synthesis of a-methyl phenylalanine has been developed based on an amidase from Pseudomonas fluorescens [IFO 3081] [14]. 

Later in the process this side chain is removed enzymatically, using an enzyme quite similar to the glutaryl amidase from Pseudomonas sp. as in the enzymatic production of 7-ACA. For the production of 7-ADCA and the dicarboxylic acid amidase, new plants are currently under construction at DSM in The Netherlands. Compared with the old process for the production of 7-ADCA, the major advantages of this process are higher purity of the end product, much greater energy efficiency and almost complete absence of organic solvents. 

Production of 7-aminocephalosporanic acid (7-ACA) (and of 7-aminodesacetoxy cepha-losporanic acid (7-ADCA)) from cephalosporins by the action of cephalosporin C amidase from Pseudomonas sp. (Toyo Jozo Inc., Asahi Chemical Industry Co., Ltd., and others). Annual world production of 7-ACA 1000 tons and 7-ADCA 500 tons, used for the manufacture of semisynthetic cephalosporins. 

Figure 19 Preparation of (5)-2-isopropyl-4-chlorophenylacetie acid by the combined action of both stereospecific nitrile hydratase and amidase from Pseudomonas sp. B21C9.

Figure 29 Racemic resolutions catalyzed by a stereospecific amidase from Pseudomonas chloraphis B23.

The processes are based on whole bacterial cells. In the case of the pipecolic acid, an important building block for pharmaceutical chemistry, an S-selective amidase in Pseudomonas fluorescens cells, catalyses the reaction with high selectivity and the acid is obtained with an ee >99% (Scheme 6.27A). For the preparation of piperazine-2-carboxylic acid from the racemic amide a R- and a S-selective amidase are available. Utilising Klebsiella terrigena cells the S-enantiomer is prepared with 42% isolated yield and ee > 99%, while Burkholderia sp. cells catalyse the formation of the -enantiomer (ee=99%, Scheme 6.27 B). 

The CO—NH amide bond is relatively energy-rich and can be hydrolyzed to free carboxylic acids and ammonia, by a variety of unrelated or distantly related enzymes, called amidases. Most of amidases are sulfhydryl enzymes like all members of the nitrilase superfamily, while other amidases such as those from Pseudomonas... 

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)-mandelic acid using S-selective hydroxynitriie iyase (HNL) from Manihot escu-lenta, nonseiective nitriiase from Pseudomonas fluorescens EBC 191, and amidase from Rhodococcus erythropolis [55]. 

L-a-Amino acids have been prepared by the resolution of racemic a-amino acid amide by the L-specific aminopeptidase from Pseudomonas putida ATCC 12633 [7]. Enzyme from R putida ATCC 12633 cannot be used to resolve a-alkyl-substituted amino acid amides 103. Aminoamidase from Mycobacterium neoaurum ATCC 25795 has been used in the preparation of L-a-alkyl amino acid 104 (Fig. 34) and D-amide of a-alkyl-substituted amino acids by enzjmaatic resolution process using racemic a-alkyl amino acid amide as a substrate [169,179]. Amidase from Ochrobactrum anthropi catalyzed the resolution of a,a-disubsituted amino acids, iV-hydroxy amino acids, and a-hydroxy acid amides. The resolution process could lead to the production of chiral amino acids or amides in 50% yield. Recently, amino acid racemases have been used to get 100% yield of chiral amino acids [179]. 

Together with R. rhodochrous ATCC 21197 [43] and Pseudomonas putida NRRL 18668 [51], also Rhodococcus sp. C3II md Rhodococcus erythropolis MP 50 were used for the enantiospecific preparation of (S)-naproxen [65]. Rhodococcus sp. C3II lacks a nitrilase but exhibits nitrile hydratase and amidase activities, both of which are constitutive and prefer the (5 )-enantiomers of naproxen derivatives. On the other hand, the enzymes from R. erythropolis MP 50 were induced by nitriles and its nitrile hydratase was (R)-specific [44]. Due to the presence of a strictly (5)-specific amidase, both strains finally formed (5)-naproxen with high enantioselectivity (Fig. 18). Evidence for the enantioselectivity of the nitrile hydratases of both strains was obtained by the formation of optically active amides in the presence of the amidase inhibitor diethyl phosphoramidate [63,65]. The nitrile hydratase of Rhodococcus sp. C3II whole cells was used for the sjmthesis of (>S)-naproxen amide with 94% e.e. after 30% conversion in the presence of the amidase inhibitor [63]. In addition, the highly stereoselective amidases of these two strains were used to prepare (5)-ketoprofen (Fig. 28), and the amidase from R. erythropolis MP 50 was used to prepare (5)-2-phenylpropionic acid with more than 99% e.e. and more than 49% conversion [66,67]. 

Figure 22 Fonnation of L-a-amino acids by L-selective amidases from Brevibacterium sp. R312 and a Pseudomonas putida strain.

L-Amino adds could be produced from D,L-aminonitriles with 50% conversion using Pseudomonas putida and Brembacterium sp respectively, the remainder being the corresponding D-amino add amide. However, this does not prove the presence of a stereoselective nitrilase. It is more likely that the nitrile hydratase converts the D,L-nitrile into the D,L-amino add amide, where upon a L-spedfic amidase converts the amide further into 50% L-amino add and 50% D-amino add amide. In this respect the method has no real advantage over the process of using a stereospecific L-aminopeptidase (vide supra). 

Novo, C., Farnaud, S., Tata, R., et al. 2002. Support for a three-dimensional structure predicting a Cys-Glu-Lys catalytic triad for Pseudomonas aeruginosa amidase comes from site-directed mutagenesis and mutations altering substrate specificity. Biochemistry Journal, 365 731-8. 

Lonza AG has reported on the use of enantioselective amidases for the resolution of piperazine-2-carboxamide and piperidine-2-carboxamide using whole cell biocatalysts from Klebsiella terrigena, Pseudomonas fluorescence and Burkholderia sp., the last containing an (R)-selective amidase (Scheme 12.2-7)[l 1. Furthermore, several amidases exhibiting high selectivities [either (Sj- or (R)-] towards 2-arylpropiona-... 

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