Stereoselective amidases

Dimethylcyclopropanecarboxylic acid is an intermediate for the synthesis cilastatin, a dehydropeptidase I inhibitor. Lonza has developed an industrial two-step biotransformation process starting from racemic 2,2-dimethylcyclopropane nitrile (34 Scheme 18). The stereoselective amidase has been found in Comomo-nas acidovorans and cloned into a fast-growing E. coli strain which produces large amounts of biomass [92, 93]. 

In a number of gram-positive organisms the combination of a stereoselective nitrile hydratase and a stereoselective amidase has been described (see chapter below). However, in the case of the acetonitrile-utilizing Rhodococcus sp. AJ 270 no amidase activity has been detected [55]. The wide-spectrum nitrile hydratase of this microorganism was used to prepare (/ )-2-phenylbutyramide from the racemic nitrile without acid as byproduct (Fig, 14). 

V. STEREOSELECTIVE NITRILE HYDRATASES IN COMBINATION WITH STEREOSELECTIVE AMIDASES... 

Among stereoselective nitrile-converting enzymes, the combined action of a stereoselective nitrile hydratase and a stereoselective amidase has been often described, and stereospecific nitrile conversions by whole cells are frequently found in the patent literature [59,60] however, careful analysis has usually revealed stereoselectivity in the amidase and not or only to a low extent in the hydratase [61,62]. If both enzymes were stereosj ific, nitrile hydratase and amidase have been described to act either synergistically or antagonistically regarding their enantioselectivity. 

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 18 Stereospecific formation of the anti-inflammatory agent (S)-naproxen by the stereoselective amidase/nitrile hydratase system from two Rhodococcus strains.

From a screening using racemic iV-heterocyclic carboxamides as sole sources of nitrogen, three strains bearing stereoselective amidases were isolated and used for the... 

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). 

Hydrolyses Esters and Amides. The plasma, liver, kidney, and intestines contain a wide variety of nonspecific amidases and esterases. These catalyze the metabolism of esters and amides, ultimately leading to the formation of amines, alcohols, and carboxylic acids. Kinetically, amide hydrolysis is much slower than ester hydrolysis. These hydrolyses may exhibit stereoselectivity. 

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). 

Strategic importance of biocatalyzed synthetic transformations in terms of eco-compatibility and cheaper processes has been widely stressed previously. Among the developed biotransformations catalyzed by nitrilases or nitrile hydratases/ amidases systems, a special interest is focused toward stereoselective reactions able to give access to molecules otherwise impossible to obtain by classical chemical routes. Hereby, selected examples aim to offer an overview of research in this direction. Examples of industrial processes using nitrile hydrolyzing biocatalysts are also illustrated. 

Despite the fact that early experiments suggested low selectivity of nitrile-converting enzymes with respect to the substrate chirality (Faber, 1992), many recent works report the successful enantioselective bioconversion of nitriles catalyzed by nitrilases or nitrile hydratases, even if the stereoselectivity of nitrile hydratases remains often lower that that of coupled amidases. 

Nitrile Hydratase/Amidase Catalyzed Stereoselective Transformation of cis- and frans-N-Protected-P-amino-cyclopentane/ hexane Nitriles... 

During our longstanding interest in the biohydrolysis of nitriles, we found that whole cell preparations of certain Rhodococci, such as R. erythropolis A4 (formerly R. equi A4), R. sp. R312, and R. erythropolis NCIMB 11540, containing the nitrile hydratase/amidase enzyme system, are efficient catalysts for stereoselective microbial hydrolysis of N-protected carbocyclic P-amino nitriles ( )-la-( )-4a, to P-amino acids lc-4c and amides lb-4b, respectively (Scheme 15.1) [33, 34]. 

In contrast to the various fermentation and bioconversion approaches that have used enzyme stereoselectivity to syntlresize L-phenylalanine as a single isomer, additional methods have been developed that rely on the same stereoselectivity to resolve racemic mixtures of chemically synthesized amino acids. One such general approach, which has been successfully commercialized for L-phenylalanine production, relies on cleavage of the L-isomer of a D,L-a-amino acid amide mixture by an enantiospecific amidase enzyme. The general procedure operated... 

The conversion of amino add amides into chiral amino acids has been the subject of a large number of monographs and reviews 21 29L In this section information will be given on amidases and aminopeptidases that have been reported for the stereoselective hydrolysis of amino acid amides. 

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). 

Polhuijs, M. Gasinska, I. Cherry, W.F. Mulder, G.J. Pang, K.S. Stereoselectivity in glutathione conjugation and amidase-catalyzed hydrolysis of the 2-bromoisovalerylurea enantiomers in the single-pass perfused rat liver. J. Pharmacol. Exp. Ther. 1993, 265, 1406 1412. 

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