Nitrilases dynamic kinetic resolution

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. 

Figure 6.30 Nitrilase-catalyzed dynamic kinetic resolution of cyanohydrins.

Nitrilase l-Catalyzed Production of Mandelic Acid Derivatives Under Dynamic Kinetic Resolution Conditions (Scheme 20.1)... 

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-... 

Scheme 5.16 The industrial synthesis of R-mandelic acid proceeds via a base and nitrilase-catalyzed dynamic kinetic resolution.

Although nitrilases do not always display high enantioselectivities [103] several examples of enantioselective nitrilases are known. Indeed they are used industrially for the synthesis of (R)-mandelic acid [34] and S-selective enzymes are also known [104]. In both cases the nitrilases were used for dynamic kinetic resolutions and they are discussed in Chapter 5 (Schemes 5.16 and 5.17). 

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]. 

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).

An enantioselective nitrilase has also been shown to be applicable in the dynamic kinetic resolution of mandelonitrile. Using the nitrilase produced by Alcaligenes faecalis ATCC 8750 Yamamoto et al. showed that they could derive (Rj-(-)-mandelic acid from mandelonitrile in 91% yield with an ee of 100%. Under the reaction conditions used non-reacting (S) -mandelonitrile undergoes spontaneous racemiza-tion leading to the high yield (see Scheme 12.1-8)[48]. Currently (R)-mandelic acid and (R)-chloromandelic acid are produced using nitrilases on an industrial scale by the Mitsubishi Rayon Corp. 

The product of a NHase/amidase cascade reaction is an acid, which is the same as the single enzymatic reaction performed by a nitrilase. However, the NHases usually have different substrate specificities than nitrilases, making them more suitable for the production of certain compounds. Although most organisms have both NHase and amidase activity (see earlier text), it is sometimes preferable, in a synthetic application, to combine enzymes from different organisms. The reasons for this are the enantioselectivity of the amidase or specific activity or substrate specificity of either of the enzymes. In this way, products with different enantiomeric purity can be obtained. Recently, a coupling of a NHase with two different amidases with opposite enantiopreference together with an -amino-a-caprolactam racemase that allows the formation of small aliphatic almost enantiopure (R)- or (S)-amino acids via dynamic kinetic resolution processes has been described [52]. 

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. 

Synthesis of (8)-mandelic acid via dynamic kinetic resolution of mandelonitrile with a wild-type whole-cell catalyst from Alcaligenes faecalis containing a nitrilase. 

Use of Nitrilases Nitrilases, or nitrile hydra-tases, catalyze the irreversible hydrolysis of nitriles into carboxylic acids, respectively, carboxylic amides, with high enantioselectivities. Therefore, they represent a good tool as a catalyst for the dynamic kinetic resolution... 

SCHEME 28.8. Nitrilase-catalyzed synthesis of carboxylic acids, based on a dynamic kinetic resolution. 

General procedure Dynamic kinetic resolution for the synthesis of (R)-mandelic acid 14 In 10 mL of Tris-HCl buffer (0.1 M, pH 7), immobilized or free cells of Pseudomonas putida MTCC 5110 (harboring a stereoselective nitrilase) at 50mg/mL and racemic mandelonitrile 2 (15 mM) were introduced. The solution was stirred for 4 hours at 35 and then stopped by addition of 1-N HCl (0.3 mL). The enzyme was filtered off and the filtrate neutralized to pH 8.5 with 2-N NaOH, washed with diethylether, readjusted to pH 1.5 with 6-N HCl, extracted with diethylether, and finally concentrated. Yield (free) %%, (immobilized cells) 93% ee 98.8%. 

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