Screening nitrile-selective

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... 

Because in methanol crystallization of amino nitrile 3 did not take place, first the solvent was varied in order to attempt to find conditions for a crystallization-induced asymmetric transformation. At a MeOH/2-PrOH ratio of 1/9, the amino nitrile (R,S)-3 was isolated in 51% yield and dr 99/1 (entry 2). Other combinations of alcoholic solvents failed to lead to a higher yield of precipitated (R,S)-3 in high dr (entries 3 and 4). On further screening of solvents, it was observed that upon addition of HjO to the methanol solution selective precipitation of amino nitrile (R,S)-3 occurred giving (R,S)-3 and (R,R)-3 in a ratio of 81 19 and 69% yield (entry 5). The asymmetric Strecker reaction was further studied in HjO alone using temperature as a variable. The results of these experiments are given in Table 1 (entries 6-9). After addition of NaCN/AcOH at 23-28 °C... 

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%). 

Martinkova, L., Vejvoda, V., and Kren, V. 2008. Selection and screening for enzymes of nitrile metabolism. Journal of Biotechnology, 133 318-26. 

Patterson, A. W., Wood, W. J. L., Hornsby, M., Lesley, S., Spraggon, G., Ellman, J. A. Identification of selective, nonpeptidic nitrile inhibitors of cathepsin S using the substrate activity screening method. J. Med. Chem. 2006,49, 6298-6307. 

N-l acetyl derivative. A much more powerful synthesis of the tert-butylamides was the Ritter reaction of the corresponding nitrile, which allowed direct production of N-l-acylated tetrahydropyrazines [18]. These in turn could be selectively further functionalized at N-4 so that selective protective group removal was possible, giving access to the final N-4 Boc substitution pattern [2], Some examples of the results obtained in the original screening are given in Tab. 7. 

Recently, metagenomic library screening provided a few more nitrilases, whose genes were obtained from clones growing on cinnamonitrile or a nitrile mixture. One of the enzymes was selected for the monohydrolysis of 2-methylglutaronitrile into... 

In contrast, the nitrile hydratase of R. rhodochrous IFO 15564, which was isolated by a screening using 3-hydroxypropionitrile and benzonitrile as the sole sources of nitrogen, was found to be (5)-selective toward substituted 3-hydroxyglutaronitriles [70]. Together with an (5)-specific amidase, it converted 3-benzoyloxyglutarodinitrile to the sole product (S)-cyanocarboxylic acid in an optically pure form without leaving any intermediate ( )-cyano amide (Fig. 21). 

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