Nitrilases oxynitrilases

Chapters 5-8 are directed to emerging enzymes, which include oxynitrilases, aldolases, ketoreductases, oxidases, nitrile hydratases, and nitrilases, and their recent applications especially in synthesis of chiral drugs and intermediates. 

Figure 8.12 Conversion of benzaldehyde into enantiomerically pure (S)-mandelic acid by the sequential addition of HCN catalyzed by the (.S )-selective oxynitrilase from Manihot esculenta (MeHnL), and subsequent hydrolysis of the resultant (5)-mandelonitrile by the nitrilase from Pseudomonas fluorescens ECB 191 (PfNLase)...

Mateo, C., Chmura, A., Rustler, S. et al. (2006) Synthesis of enantiomerically pure (S)-mandelic acid using an oxynitrilase-nitrilase bienzymatic cascade a nitrilase surprisingly shows nitrile hydratase activity. Tetrahedron Asymmetry, 17, 320-323. 

In basic chemicals, nitrile hydratase and nitrilases have been most successful. Acrylamide from acrylonitrile is now a 30 000 tpy process. In a product tree starting from the addition of HCN to butadiene, nicotinamide (from 3-cyanopyridine, for animal feed), 5-cyanovaleramide (from adiponitrile, for herbicide precursor), and 4-cyanopentanoic acid (from 2-methylglutaronitrile, for l,5-dimethyl-2-piperidone solvent) have been developed. Both the enantioselective addition of HCN to aldehydes with oxynitrilase and the dihydroxylation of substituted benzenes with toluene (or naphthalene) dioxygenase, which are far superior to chemical routes, open up pathways to amino and hydroxy acids, amino alcohols, and diamines in the first case and alkaloids, prostaglandins, and carbohydrate derivatives in the second case. 

FIGURE 17.7 Bienzymatic oxynitrilase/nitrilase synthesis of (5)-mandelic acid. 

Figure 16.1 Synthetic routes to enantiomerically pure 2-hydroxycarboxylic acids, via oxynitrilase (hydroxynitrile lyase) catalysed enantioselective hydrocyanation (route A) and (R)-nitrilase (nitrilase) mediated dynamic kinetic resolution (route B).

The nitrilase mediated DKR route to enantiomerically pure 2-hydroxycarboxylic acids is restricted to the (R)-enantiomers because, to our knowledge, no (S)-selec-tive nitrilases for cyanohydrin substrates are commonly available [11]. We reasoned that a fully enzymatic route to the (S)-acids should be possible by combining an (S)-selective oxynitrilase (hydroxynitrile lyase, EC 4.1.2.10, (S)-hydroxynitrile lyase) and a non-selective nitrilase in a bienzymatic cascade (see Figure 16.3). Besides being more environmentally acceptable than chemical hydrolysis, the mild reaction conditions of the combined enzymatic reaction would be compatible with a wide range of hydrolysable groups. 

The bienzymatic transformation of aldehydes and HCN in the presence of an oxynitrilase and a nitrilase is a useful addition to the synthetic repertoire, provided that the coproduction of amide can be avoided. 

However, this route was not developed further because of the amount and resulting cost of the enzyme required to complete the reaction in a reasonable time. Other possible routes with one or more biocatalytic steps included those involving an enantioselective oxynitrilase reaction (Fig. 6). According to the choice of enzyme, it could be possible to form either the (R)- or the (S)-enantiomer. Fig. 7 depicts various routes starting from the racemic cyanohydrin. Nitrilases convert nitriles into the corresponding acids and are sometimes stereospecific. Nitrile hydra-tases convert nitriles into amides, and are also sometimes stereospecific. Ami-dases convert amides into the corresponding acids and are often stereospecific. Screening for enantioselective oxynitrilases [14] and for enantiospecific nitrilases [15] was started, but discontinued when the amidase route (below) was found to be successful. 

Sosedov, O., Matzer, K., Burger, S., Kiziak, C., Baum, S., Altenbuchner, J., Chmura, A., van Rantwijk, F., and Stolz, A. (2009) Construction of recombinant Escherichia coli catalysts which simultaneously express an (S)-oxynitrilase and different nitrilase variants for the synthesis of (S)-mandelic acid and (S)-mandelic amide from benzaldehyde and cyanide. Adv. Synth. Catal., 351, 1531-1538. 

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