Oxynitrilases

Cyanohydrin Synthesis. Another synthetically useful enzyme that catalyzes carbon—carbon bond formation is oxynitnlase (EC 4.1.2.10). This enzyme catalyzes the addition of cyanides to various aldehydes that may come either in the form of hydrogen cyanide or acetone cyanohydrin (152—158) (Fig. 7). The reaction constitutes a convenient route for the preparation of a-hydroxy acids and P-amino alcohols. Acetone cyanohydrin [75-86-5] can also be used as the cyanide carrier, and is considered to be superior since it does not involve hazardous gaseous HCN and also virtually eliminates the spontaneous nonenzymatic reaction. (R)-oxynitrilase accepts aromatic (97a,b), straight- (97c,e), and branched-chain aUphatic aldehydes, converting them to (R)-cyanohydrins in very good yields and high enantiomeric purity (Table 10). 

Table 10. Synthesis of Cyanohydrins by Oxynitrilase-Catalyzed Transacylation...

The apphcation of (5)-oxynitrilase has been reported only recendy (159). The enzyme isolated from shoots of Sorghum catalyzes the condensation between various 3- and 4-substituted benzaldehydes and hydrogen cyanide resulting in (5)-cyanohydrins in 80—90% yield and up to 99% ee. 

I.3.7.I.I. (/ )-Oxynitrilase-Catalyzed Addition of Hydrocyanic Acid to Aldehydes... 

As early as 1908, Rosenthaler found in the ferment mixture of emulsin a u-oxynitrilase , which directed the addition of hydrocyanic acid (hydrogen cyanide) to benzaldehyde asymmetrically to give x-hydroxybenzeneacetonitrilc (mandelonitrile)9. This result was confirmed1 °, however, it was not until 1963 that Pfeil ct al. first isolated and characterized the enzyme (R)-oxyni-trilase [EC 4.1.2.101 from bitter almonds (Prunus amygdalus)1 12. The yellow-colored enzyme contains a flavin-adenine dinucleotide (FAD)11 and loses its activity by splitting off this prosthet-... 

Method B A solution of (/ )-oxynitrilase (150 pL, sec Method A) is dropped onto 2 g of Avicel cellulose [soaked in 20 mL of 0.01 M acetate buffer (pH 5.4) for 1 2 h, filtered off and pressed]. 20 mL of ethyl acetate (saturated with 0.01 M acetate buffer, pi I 5.4) are added, followed by 5 mmol of aldehyde and 250 pL (6.5 mmol) of hydrocyanic acid. After stirring at r.t. (Tabic 1) the mixture is filtered, and the filter cake pressed and washed with ethyl acetate. The combined solutions are dried and concentrated. 

Recently, the enantioselective addition of hydrocyanic acid to aldehydes, analogous to the synthesis of (/ )-cyanohydrins, yielding (.S)-cyanohydrins in very high optical purity, with (S )-oxynitrilase as catalyst, was reported20,21. 

S )-Oxynitrilase-catalyzed reactions, as a rule, are slower than those for the comparable reactions with (R)-oxynitrilase. The use of organic solvents20 instead of aqueous systems21 is of great advantage. 

By simply hydrolyzing the easily accessible 2-hydroxy-2-methylalkanenitriles with concentrated acid, 2-hydroxy-2-methylalkanoic acids are obtained without measurable racemization (Table 3). The reaction sequence from the starting ketone to the carboxylic acid can be carried out in one pot without isolation of the cyanohydrin. The enantiomeric excesses of the (/ )-cyanohydrins and the (ft)-2-hydroxyalkanoic acids are determined from the ( + )-(/T)-Mosher ester derivatives and as methyl alkanoates by capillary GC, respectively. The most efficient catalysis by (R)-oxynitrilase is observed for the reaction of hydrocyanic acid with 2-alkanoncs. 3-Alkanoncs are also substrates for (ft)-oxynitrilase, to give the corresponding (/ )-cyanohydrins32. 

Chiral Synthesis of Pharmaceutical Intermediates Using Oxynitrilases... 

Chapters 5-8 are directed to emerging enzymes, which include oxynitrilases, aldolases, ketoreductases, oxidases, nitrile hydratases, and nitrilases, and their recent applications especially in synthesis of chiral drugs and intermediates. 

Hydroxynitrile lyases (HNLs or oxynitrilases) catalyze C—C bond-forming reactions between an aldehyde or ketone and cyanide to form enantiopure cyanohydrins (Figure 1.15), which are versatile building blocks for the chiral synthesis of amino acids, hydroxy ketones, hydroxy acids, amines and so on [68], Screening of natural sources has led to the discovery of both... 

Vorlop et al. described a novel cross-linked and subsequently poly(vinyl alcohol-entrapped PaHNL for synthesis of (//(-cyanohydrins. These immobilized lens-shaped biocatalysts have a well-defined macroscopic size in the millimeter range, show no catalyst leaching, and can be recycled efficiently. Furthermore, this immobilization method is cheap and the entrapped (/ )-oxynitrilases gave similar good results compared with those of free enzymes. The (//(-cyanohydrin was obtained in good yields and with high enantioselectivity of up to >99% ee [55],... 

Ognyanov, V.I., Datcheva, V.K. and Kyler, K.S. (1991) Preparation of chiral cyanohydrins by an oxynitrilase-mediated transcyanation. Journal of the American Chemical Society, 113, 6992-6996. 

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)...

Mateo, C., Chmura, A., Rustler, S. et al. (2006) Synthesis of enantiomerically pure (S)-mandelic acid using an oxynitrilase-nitrilase bienzymatic cascade a nitrilase surprisingly shows nitrile hydratase activity. Tetrahedron Asymmetry, 17, 320-323. 

The best results for the asymmetric cyanohydrination reactions are obtained through biocatalysis, using the readily available enzyme oxynitrilase. This provides cyanohydrins from a number of substances with over 98% ee.146... 

Oxynitrilases, isolated from either almond [(J )-specific]2° or a microorganism [( -specific],21 catalyze the enantioselective addition of cyanide ion to a range of aromatic or aliphatic aldehydes, providing cyanohydrins with ee values of up to 99%. 

Since the reaction has been reviewed recently (12) only a few additional facts will be mentioned. Many optically active cyanohydrins can be prepared (33) with e.e. s of 84 to 100% by the use of the flavopnotein D-oxynitrilase adsorbed on special (34) cellulose ion-exchange resins. Although the enzyme is stable, permitting the use of a continuously operating column, naturally only one enantiomer, usually the R isomer, is produced in excess. This (reversible) enzyme-catalyzed reaction is very rapid (34). Nonenzymic catalysts, such as the cinchona alkaloids, permit either enantiomer to be prepared in excess. 

Fechter, M.H., Gruber, K., Avi, M., Skranc, W., Schuster, C., Pbchlauer, P., Klepp, K.O. and Griengl, H., Stereoselective biocatalytic synthesis of (S)-2-hydroxy-2-methylbutyric acid via substrate engineering by using thio-disguised precursors and oxynitrilase catalysis. Chem. Eur. J., 2007,13, 3369. 

Chen, P., Han, S., Lin, G., Huang, H. and Li, Z., A study of asymmetric hydrocyanation of heteroaryl carboxaldehydes catalyzed by (i )-oxynitrilase under micro-aqueous conditions. Tetrahedron Asymm., 2001,12, 3273. 

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