Nitrilases substrate selectivity

I 14 Enzymatic Stereoselective Synthesis of fi-Amino Acids 14.3.1.2 Nitrilase Substrate Selectivity... 

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],... 

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%). 

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-... 

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). 

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. 

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 well-known classification of nitrilases into different substrate specificity sub-types [2] was demonstrated to be in partial correlation with their aa sequence similarities. A prediction of substrate specificities in putative nitrilases could be made by grouping similar sequences. For instance, four probable arylacetonitri-lases were selected because of their high similarity (over 50%) to the biochemically characterized enzyme from Neurospora crassa, and their expected substrate specificities were confirmed [6]. Using this approach, it also seems possible to predict cyanide hydratases [5,6]. Aromatic nitrilases are not as easy to predict Members of... 

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. 

The mmtiber of commercial nitrilases has recently increased considerably. Catalysts suitable for the transformation of a specific substrate can be selected from nitrilase sets available from Codexis and Prozomix Limited [10,11]. A similar set of nitrile hydratases (NHases) is also available from the latter company. 

Nitrilases suitable for the transformations of alicyclic five- and six-membered y-amino nitriles were selected from the set of commercial nitrilases available from BioCatalytics Inc. (now Codexis) [59] and the effects of the substrate structure (ring size, protecting group, and trans versus cis configuration) were studied. N-tosylated derivatives of cis-3-amino cydopentane- and cyclohexanecarboxylic acids were obtained with the highest enantiopurities. Production of N-substituted pyrrolidine-and piperidinecarboxylic acids was also possible using the same nitrilases, but the enantiopurities of these products were generally low [60]. 

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