Cyanohydrins nitrilase

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. 

Burk and coworkers have used a variety of nitrilases for the DKR of cyanohydrins [48]. Nitrilases catalyze the hydrolytic conversion of cyanohydrins directly to the corresponding carboxylic acids. Racemization was performed under basic conditions (phosphate buffer, pH 8) through reversible loss of HCN. (R)-Mandelic acid was obtained in high yield (86% yield) and high enantioselectivity (98% ee) after 3 hours (Figure 4.23). 

The addition of HCN to aldehydes or ketones produces cyanohydrins (a-hydroxy nitriles). Cyanohydrins racemize under basic conditions through reversible loss of FiCN as illustrated in Figure 6.30. Enantiopure a-hydroxy acids can be obtained via the DKR of racemic cyanohydrins in the presence of an enantioselective nitriletransforming enzyme [86-88]. Many nitrile hydratases are metalloenzymes sensitive to cyanide and a nitrilase is usually used in this biotransformation. The DKR of mandelonitrile has been extended to an industrial process for the manufacture of (R)-mandelic acid [89]. 

Figure 6.30 Nitrilase-catalyzed dynamic kinetic resolution of cyanohydrins.

One of the most attractive biocatalytic options is the nitrilase-catalysed enantioselective hydrolysis of the racemic cyanohydrin. The hydroxyacid is produced directly without need for protection/deprotection steps and cyanohydrins racemize spontaneously at neutral or... 

The group of Burk has used a variety of nitrilases for the DKR of cyanohydrins [23]. Nitrilases catalyze the hydrolytic conversion of cyanohydrins directly to the corresponding carboxylic acid. Racemization was performed under basic... 

A drawback of this reaction has recently been addressed. Only very few S-selective nitrilases were known this problem has been solved a systematic screening program yielded a number of S-selective nitrilases that have successfully been employed in this dynamic kinetic resolution (Scheme 5.17) [38]. In an alternative approach, combining the enantioselectivity of an HNL with the hydrolytic power of a not very selective nitrilase that did accept cyanohydrins as substrates, the synthesis of optically enriched a-hydroxy acids starting from alde-... 

FIGURE 17.8 Dynamic kinetic resolution of cyanohydrins catalyzed by nitrilases. 

Alternatively, enantiopure 2-hydroxycarboxylic acids can be obtained via a dynamic kinetic resolution of the (chemically synthesized) cyanohydrin in the presence of an enantioselective nitrilase (EC 3.5.5.1) (see Figure 16.1, route b). Racemization of the cyanohydrin, via reversible dehydrocyanation, takes place readily at pH 7 or above. The methodology [1] is attractive on account of the mild reaction conditions and is industrially applied in the multiton-scale synthesis of (R)-mandehc acid [2]. 

This nitrilase dynamic kinetic resolution (DKR) methodology depends on the availability of highly enantioselective biocatalysts that generate a minimum amount of amide. This latter issue may seem trivial and has long been disregarded somewhat, but reports of modest amounts of amide co-products date back to the early days of nitrilase enzymology. Only recently has the subject come under more intense scrutiny [3-5] and has a relationship with the stereochemistry of the nitrile been demonstrated [3, 5]. Hence, we set out to investigate the enantiomer and chemical selectivity of nitrilases in the hydrolysis of a representative set of cyanohydrins. 

Three cyanohydrins (la-c) (see Table 16.1) were subjected to hydrolysis in the presence of a number of nitrilases (Figure 16.2). The reactants included the standard substrate mandelonitrile (la) and its o-chloro derivative (lb), which is of... 

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. 

Commonly available nitrilases hydrolyze cyanohydrins with modest to excellent selectivity for the (R)-enantiomer. The formation of amide coproducts, once thought to be an insignificant side-reaciion, varies erratically in cyanohydrin hydrolysis between minute and copious, depending on the enzyme and the steric character of the nitrile. 

Cyanohydrins are a very useful class of compounds as they can be transformed into a wide variety of compounds while retaining the stereogenic center (32, 35). Hydroxy nitrilases are available from natural sources (13), which can give access to either enantiomer of the product cyanohydrin (Fig. 1) (47). 

Figure 1 Cyanohydrin formation with hydroxy nitrilases.

Enzymes of the hydroxynitrilase dass catalyze the addition of HCN to aldehydes, produdng cyanohydrins. Recendy, the reaction has been extended to a few ketones with modified hydroxynitrilase enzymes. In many cases, these are formed with good optical purities and such reactions are the simplest type of enzyme catalyzed carbon-carbon bond formation. By pairing hydroxynitrile lyases with nitrilases or nitrile hydratases, one-pot, multistep conversions become possible, and this also shifts the equilibrium to favor the addition products. Such concerns are particularly important when applying these catalysts to ketones where the equilibrium generally favors the starting carbonyl compound (Figure 1.17). 

Dynamic kinetic resciution of racemic cyanohydrins 25 using recombinant nitrilases toward enantiopure mandeiic and iactic acid derivatives 26 [31]. 

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]. 

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

After acetylation with vinyl acetate, the Amano lipase PS was used for the (R)-specific ester cleavage of (R,S)-cyanohydrin acetate [113]. The chirality of acetylated (JS)-and nonacetylated (JR)-cyanohydrin products was enhanced using R. rhodochrous ATCC 21197 for the further conversion to optically active a-hydroxy acids (Fig. 35), which are useful starting materials in synthetic organic chemistry [90]. The Rhodococcus strain has been reported to contain a stereospecific nitrilase (Fig. 22), a stereospecific nitrile hydratase (Figs. 10 and 11), and a stereospecific amidase (Figs. 16 and 27). 

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