Nitrile-converting enzymes nitrilases

The application of nitrile-converting enzymes, nitrilases, and hydroxynitrile lyases in the s)mthesis of chiral compounds and cyanohydrins is covered. 

As illustrated in Figure A8.3 nitrilases catalyse conversions of nitriles directly into the corresponding carboxylic adds (route A), while other nitrile converting enzymes, die nitrile hydratases, catalyse the conversion of nitriles into amides (route B) which, by the action of amidases usually present in the whole cell preparations, are readily transformed into carboxylic adds (route C). 

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. 

Previous reviews of this field of research focused mainly on the biocatal5dic uses of nitrilases [1], structure and function of nitrilases [2, 3], nitrilases in filamentous fungi [4], stereoselective biotransformations catalyzed by nitrile-converting enz5nnes [5], and the structure, function, and uses of nitrile hydratases [6]. For a comprehensive review of nitrile-converting enzymes known at the time, the study by Banerjee et al. [7] has been helpful. Some of the most recent reviews focused on nitrilases, specifically their sources, properties, and use, [8], and on methodologies for their screening [9]. 

One example of an (S)-profen-fornung enzyme is the stereoselective nitrilase from R. rhodochrous ATCC 21197 used for the conversion of racemic 2-aryl propionitriles to the corresponding (5)-acids [43,44]. The highest optical and chemical yields were obtained with 2-(4-methoxyphenyl)propionic acid (Fig. 10). In this Rhodococcus strain all three nitrile-converting enzymes have been found (Fig. 16, 21, and 27). 

High enantioselectivity has been more often described with amidases and nitrileforming enzymes than with the directly nitrile-converting nitrilases and nitrile hydratases. This might indicate a lower stereoselective capacity of directly nitrile-converting enzymes but is probably also in part due to the fact that the stereospecificity of these enzymes has only been the subject of intense study in the last decade. 

A strategy to access lactones via enzymatic hydrolysis of y- and /3-hydroxy aliphatic nitriles to their corresponding acids with subsequent internal esterification was applied using commercially available enzymes from BioCatalytics Inc. A number of y- and /3-hydroxy aliphatic nitrile substrates (Table 8.11) were evaluated, with the greatest selectivity observed with y-hydroxy nonanitrile, which was converted by nitrilase NIT1003 to the precursor of the rice weevil pheromone in 30% yield, 88% ee with an enatiomeric ratio of = 23 [90],... 

Nitrilase [EC 3.5.5.1], also known as nitrile aminohy-drolase and nitrile hydratase, catalyzes the hydrolysis of a nitrile to produce a carboxylate and ammonia. The enzyme acts on a wide range of aromatic nitriles. Nitrile hydratase [EC 4.2.1.84], also known as nitrilase, catalyzes the hydrolysis of a nitrile to produce an aliphatic amide. The enzyme acts on short-chain aliphatic nitriles, converting them into the corresponding acid amides. However, this particular enzyme does not further hydrolyze these amide products nor does the enzyme act on aromatic nitriles. 

Notably, nitrile-degrading enzymes (e.g. nitrilase that converts the CN group to carboxylic acid, and nitrile hydratase that produces an amide function) have been described, and they co-exist with aldoxime-degrading enzymes in bacteria (Reference 111 and references cited therein). Smdies in this area led to the proposal that the aldoxime-nitrile pathway, which is implemented in synthesis of drugs and fine chemicals, occurs as a natural enzymic pathway. It is of interest that the enzyme responsible for bacterial conversion of Af-hydroxy-L-phenylalanine to phenacetylaldoxime, an oxidative decarboxylation reaction, lacks heme or flavin groups which are found in plant or human enzymes that catalyze the same reaction. Its dependency on pyridoxal phosphate raised the possibility that similar systems may also be present in plants . 

There are two distinct classes of enzymes that hydrolyze nitriles. Nitrilases (EC. 3.5.5.1) hydrolyze nitriles directly to corresponding acids and ammonia without forming the amide. In fact, amides are not substrates for these enzymes. Nitriles also may be first hydrated by nitrile hydratases to yield amides which are then converted to carboxylic acid with amidases. This is u two-enzyme process, in which enanlioselectivity is generally exhibited by the amidase. rather than the hydratase. 

The nitrile group is a versatile building block, in particular since it can be converted into acids or amides. It undergoes hydrolysis but requires relatively harsh reaction conditions. Nature provides two enzymatic pathways for the hydrolysis of nitriles. The nitrilases convert nitriles directly into acids, while the nitrile hy-dratases release amides. These amides can then be hydrolysed by amidases (see also above). Often nitrile hydratases are combined with amidases in one host and a nitrile hydratase plus amidase activity can therefore be mistaken as the activity of a nitrilase (Scheme 6.32). A large variety of different nitrilases and nitrile hydratases are available [100, 101] and both types of enzyme have been used in industry [34, 38, 94]. 

The enzymatic hydrolysis of a broad range of nitriles to the corresponding amides and acids is documented [35]. These conversions are effected directly by nitrilases or by successive action of a nitrile hydratase and an amidase. Most of these enzymes are usually unstable and whole-cell preparations are preferred. However, recently a purified nitrile hydratase preparation without amidase activity was shown to convert several 2-arylpropionitriles enantioselectively to the corresponding optically active amides (eq. (3)) [36]. 

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. 

The two enzyme classes nitrile hydratases (RCN + H20 — RCONH2) and nitrilases (RCN + 2H20 —y RCOOH + NH3) actually belong to two distant groups in the EC system, with the hydratases being classified as lyases (EC 4.2.1.84) and nitrilases as hydrolases (EC 3.5.5.1). Microorganisms that produce a nitrile hydratase also seem to produce amidases, which enable them to convert nitriles into carboxylic acids in a two-step reaction. Actually, amidase side-activity can be a problem with commercial nitrile hydratase preparations (if the target structure is the amide). Nitrilases, however, hydrolyze the nitrile without the formation of a free amide intermediate. 

The nitrilase from P. Jluorescms EBC191 converts certain nitriles not only to the acids but also forms significant amounts of the corresponding amides [55] and recently several enzyme variants have been constructed that form significantly increased amounts of amides from nitriles [70, 71]. These nitrilase variants in combination with enantioselective HnLs also offer the possibility to synthesize chiral 2-hydroxyamides from aldehydes (and ketones) and cyanide [66]. 

The immobilized enzyme preparation SP409", possessing both nitrilase and amidase activity, has been used for the hydrolysis of glycosyl cyanides. The a-nitrile 20 underwent hydrolysis to the amide 21, whilst the corre nding P-anomer was converted, at a faster rate, into the analogous carboxylic acid. ... 

The stereospecific nitrilase of this strain was also able to convert naproxen nitrile to (5 )-naproxen [(5 )-2-(6-methoxy-2-napthyl)propionic acid]. However, a high enantioselec-tivity was only found at the expense of a low chemical yield (Fig. 11). (S)-Naproxen was furthermore synthesized by stereospecific nitrile hydratase/amidase enzyme systems from oth i Rhodococcus strains (Fig. 18). 

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