Nitrilase selectivity

Many nitrilases have been screened for their abihty to catalyze enantioselective production of valuable carboxybc acid analogs with substitution in the a, p, or y position. For instance, nitrilase selectivity profiles identified a catalyst capable of resolution of racemic mandelonitrile (an a-hydroxynitrile). Strains such as an Alcali-genes sp. [44], Pseudomonas putida, and Microbacterium paraoxydans [45] showed >93% ee when hydrolyzing mandelonitrile to (J )-mandelic acid (Table 14.1). In another example, researchers at Diversa reported on the enantioselective conversion of aromatic aminonitrile compounds for the production of a-amino acids [46]. The authors were successful in achieving a 79.5% yield of the (R)-acid product at an enantiomeric excess of 96.3% from the substrate 4-fluorophenylglydnonitrile. 

Hie bioconversion of a-aminonitriles, although up until now not used on an industrial scale, is of practical interest in the production of optical active a-amino adds. This, however, will only be the case if one can select a nitrilase that enantioselectively hydrolyses die aminonitrile. 

Upon screening genomic libraries obtained from environmental samples, more than 200 new nitrilases that allow mild and selective hydrolysis of nitriles were discovered [56]. One of them catalyzes the (R)-selective hydrolysis of (16) with a value... 

The nitrilase from a nnmber of strains of Pseudomonas sp. mediated an enantiomerically selective hydrolysis of racemic 0-acetylmandelonitrile to o-acetytmandelic acid R-( )-acetylmandelic acid (Layh et al. 1992). 

Nitrilases convert nitriles to the corresponding carboxylic acids and NH3 through a cysteine residue in the active site [50]. Because of their high enantio- and regio-selectivity, nitrilases are attractive as green catalysts for the synthesis of a variety of carboxylic acids and derivatives (Figure 1.10) [51,52]. Recently, a number of recombinant nitrilases have been cloned and characterized heterologously for synthetic applications [50,53,54]. 

Similarly, the selective herbicides, bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) and ioxynil (3,5-diiodo-4-hydroxybenzonitrile) are degraded by soil bacteria to their corresponding amide products 3,5-dibromo-4-hydroxybenzamide (BrAM) and 3,5-diiodo-4-hydroxybenzamide (IAM) but are not further degraded to the corresponding acids. The identification of amidases or nitrilases able to effect these transformations, in a soil bacterium, would be of value as a bioremediation agent [48],... 

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)...

The recombinantly expressed nitrilase from Pseudomonas fluorescens EBC 191 (PFNLase) was applied in a study aimed at understanding the selectivity for amide versus acid formation from a series of substituted 2-phenylacetonitriles, including a-methyl, a-chloro, a-hydroxy and a-acetoxy derivatives. Amide formation increased when the a-substituent was electron deficient and was also affected by chirality of the a- stereogenic center for example, 2-chloro-2-phenylacetonitrile afforded 89% amide while mandelonitrile afforded 11% amide from the (R)-enantiomer but 55% amide was formed from the (5)-enantiomer. Relative amounts of amide and carboxylic acid was also subject to pH and temperature effects [87,88]. 

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],... 

Figure 8.14 Preparation of (S)-3-cyano-5-methyl hexanoic acid from isobutylsuccinonitrile using a regio- and stereo-selective nitrilase from AtNitl Arabidopsis thaliana...

A prochiral bis(cyanomethyl) sulfoxide was converted into the corresponding mono-acid with enantiomeric excesses as high as 99% using a nitrilase-NHase biocatalyst. The whole-cell biocatalyst Rhodococcus erythropolis NCIMB 11540 and a series of commercially available nitrilases NIT-101 to NIT-107 were evaluated in this study. As outlined in Figure 8.18, the prochiral sulfoxide may be transformed into five different products (plus enantiomeric isoforms), of which, three are chiral (A, B, and C) and two achiral (D and E). Only products A, B, and E were observed with the biocatalysts employed in this investigation. Both enantiomerically enriched forms of both A and C could be obtained with one of the catalysts used. The best selectivities are as follows (S)-A 99% ee, (R)-A 33% ee, (S)-C 66% ee, and (R)-C 99% ee, using NIT-104, NIT-103, NIT-108, and NIT-107 respectively. Each of these catalysts produced more... 

Zhu, D., Mukherjee, C., Biehl, E.R. and Hua, L. (2007) Nitrilase-catalyzed selective hydrolysis of dinitriles and green access to the cyanocarboxylic acids of pharmaceutical importance. AdviOU cd Synthesis and Catalysis, 349, 1667-1670. 

This screening system has also been applied successfully in the directed evolution of enantioselective EHs acting as catalysts in the kinetic resolution of chiral epoxides 95,96) (Section IV.A.4). Moreover, the firm Diversa has applied the MS-based method in the desymmetrization of a prochiral dinitrile (l,3-dicyano-2-hydroxypropane) catalyzed by mutant nitrilases 46). In this industrial application, one of the nitrile moieties was labeled selectively with as in N-17, which means that the two pseiido-eaaniiovaenc products (S)- N-18 and (J )-18 differ by one mass unit. This is sufficient for the MS system to distinguish between the two products quantitatively 46). 

In the screening of genomic libraries prepared from environmental samples collected in various parts of the world, more than 200 new nitrilases were discovered that allow mild and selective hydrolysis of nitriles (150). One of them catalyzes the (J )-selective hydrolysis of 35 with an ee value of 94.5% at a substrate concentration of 100 mM (46). However, when experiments were done at a more practical concentration of 2.25 M, activity and enantioselectivity suffered (ee only 87.8%). 

Therefore, directed evolution was applied to solve these problems. To screen for enantioselectivity, the Miilheim MS-based high-throughput ee assay (92,93) (Section III.C) was applied (46). In this case, the necessary isotope labeling focused on the use of in the pseudo-meso compound N-(J )-17 (see Section III.C for a detailed discussion). An (5)-selective nitrilase leads preferentially to N-(5)-18, whereas an 7 -selective variant results in the picw o-enantiomer (J )-18. They differ by one mass unit and can therefore be distinguished by MS, both qualitatively and quantitatively (by integration of the relevant peaks). 

A crude mixture of enzymes isolated from Rhodococcus sp. is used for selective hydrolysis of aromatic and aliphatic nitriles and dinitriles (117). Nitrilase accepts a wide range of substrates (Table 8). Even though many of them have low solubility in water, such as (88), the yields are in the range of 90%. Carboxylic esters are not susceptible to the hydrolysis by the enzyme so that only the cyano group of (89) is hydrolyzed. This mode of selectivity is opposite to that observed upon the chemical hydrolysis at alkaline pH, esters are more labile than nitriles. Dinitriles (90,91) can be hydrolyzed regioselectively resulting in cyanoacids in 71—91% yield. Hydrolysis of (92) proceeds via the formation of racemic amide which is then hydrolyzed to the acid in 95% ee (118). Prochiral 3-substituted glutaronitriles (93) are hydrolyzed by Phodococcus butanica in up to 71% yield with excellent selectivity (119). 

Nature provides a vast variety of -selective nitrilases that accept aliphatic and aromatic substrates. This disconnection can therefore be applied for the synthesis of many structurally different a-hydroxy acids. Industrially the process is applied on a multi-ton scale, to prepare (R)-mandelic acid and its analogs (Scheme 5.16) [9]. What makes the process particularly interesting from a green point of view, is that no organic solvents are used and the reaction is performed at ambient temperatures. 

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-... 

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). 

Nitrilases have been utilized successfully for the desymmetrisation of symmetric starting materials. At Diversa it was demonstrated that mutagenesis could create a highly selective nitrilase that was active at high substrate concentrations [105]. For their enzymatic route towards the atorvastatin (lipitor) side-chain this nitrilase now converts a symmetric precursor with 600 g IT1 d-1 into the enan-tiopure (R)-4-cyano-3-hydroxybutyric acid (ee=98.5%, Scheme 6.34). 

Selective nitrilases have also been developed for the enantiopure preparation of ibuprofen [106]. In a kinetic resolution with Acinetobacter sp. AK226 (S)-ibu-profen could be prepared in good optical purity (Scheme 6.35). 

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. 

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. 

Effenberger, F. and Oswald, S. 2001b. (i )-Selective hydrolysis of (ii,Z)-a,P-unsaturated nitriles by the recombinant nitrilase AtNITl from Arabidopsis thaliana. Tetrahedron ... 

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