Nitrilase-catalyzed transformations

The initial steps of nitrilase-catalyzed transformations of nitriles were proposed to consist in (i) the formation of thioimidate originating from the nucleophilic attack of the catalytic cysteine on the cyano group carbon atom and (ii) the addition of a water molecule to the thioimidate to form a tetrahedral intermediate. The formation of two different products is probably caused by two alternative cleavage pathways of this tetrahedral intermediate, either into the acylenzyme and ammonia or to the free enzyme and amide [3] (Figure 12.2). The ratio of the products is... 

Figure 12.2 Hypothetical mechanism of the nitrilase-catalyzed transformation of nitriles into carboxylic acids or amides. (According to [3].) E-SH enzyme with a cysteine as catalytic nucleophile.

Nitrilase Catalyzed Stereoselective Transformation of cis- and trans-N-Protected Conformationally Constrained y-Amino Butyronitriles... 

There are many examples of nitrilase-catalyzed reactions in which amides form a considerable amount of the reaction products, such as the transformations of acrylonitrile analogs and a-fluoroarylacetonitriles by nitrilase 1 from Arabidopsis thaliana [17], the conversion of p-cyano-L-alanine into a mixture of L-asparagine and L-aspartic acid by nitrilase 4 from the same organism [18] or the transformations of mandelonitrile by nitrilase from Pseudomonas jhiorescens [19] or some fungi [8], Moreover, formamide is the only product of the cyanide transformation by cyanide hydratase. Therefore, this enzyme was classified as a lyase (EC 4.2.1.66), although it is closely related to nitrilases, as far as its aa sequence and reaction mechanism are concerned [3]. 

The enzyme mentioned above from B. japonicum also transformed p-hydroxynitriles into acids but with low enantioselectivities. This problem was circumvented by using optically pure p-hydroxynitriles as substrates, which were prepared by the enantiose-lective reduction of p-ketonitriles catalyzed by carbonyl reductase [58]. The advantage of the nitrilase-catalyzed step was its mild and stereoretentive conditions. In this way, various substituted 3-aryl-3-hydroxypropanoic acids were prepared in both enantiopure forms (Figure 12.5). 

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. 

Nitrile biodegradation is performed by a variety of microorganisms and proceeds through two different enzymatic pathways direct transformation to carboxylic acids and ammonia, with some exceptions, catalyzed by a nitrilase (EC 3.5.5.1) [1-3] or a two-step reaction, the first catalyzed by nitrile hydratase (EC 4.2.1.84) that produces an amide intermediate, which is further hydrolyzed to the acid and ammonia by an amidase (EC 3.5.1.4) [4, 5],... 

The NHase/amidase in R. erythropolis A4, a strain used to hydrolyze a wide spectrum of nitriles [16], was recently applied to the biotransformation of benzonitrile analogs used as herbicides (Figure 11.4) and the products and parent compounds were compared in terms of their acute toxicides [17]. In other rhodococcal strains, the same compounds, apart from dichlobenil, can also be hydrolyzed in a direct pathway catalyzed by a nitrilase [18,19]. It was demonstrated that the hydrolysis of the nitriles cannot itself be considered a detoxification. The two-step transformation may be especially important in the natural degradation of these compounds because unlike nitrilases, NHases and amidases are often constitutive enzymes, and their producer strains form the typical constituents of soil microflora [17, 20]. 

Enantioselective transformations catalyzed by nitrilases often suffer from poor chiral recognition. Exceptions from this trend are benzaldehyde and phenylac-etaldehyde cyanohydrins. As an additional advantage, these substrates racemize readily at near-neutral pH via reversible loss of hydrogen cyanide representing good starting materials for dynamic kinetic resolution processes. This was demonstrated using 22 substituted phenyl and heteroaryl derivates 25 with two recombinant nitrilases a preparative biotransformation yielded (S)-phenyllactic add 26 in 84% yield and 96% ee on 1 g scale (Scheme 9.7) [31]. 

A further hydrolytic process in which whole-cell catalysis turned out to be very suitable is the transformation of a racemic nitrile into the corresponding acid exemplified for the dynamic kinetic resolution of mandelonitrile into (R)-mandelic acid, (R)-IO. This reaction is catalyzed by means of a nitrilase, which is known as highly enantioselective enzyme. As early as 1991, researchers from Asahi Chemical Industry Ltd. reported such a reaction utilizing wild-type whole cells from Alcaligenes faeccdis bearing a suitable nitrilase [32]. When starting from racemic mandelonitrile, rac-7. 

Ibuprofen [(iS)-2-(4-isobutylphenyl)propionic acid] has been prepared by resting cells of Acinetobacter sp. AK 226 [34]. Evidence for a single-step nitiilase-catalyzed reaction was obtained by the lack of an intermediate amide formation, the remaining R) nitrile (Fig. 12), and the inability to transform the racemic amide. The (S)-specific nitrilase was purified and characterized as a typical high molecular mass enzyme of 580 kDa [45]. 

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