Hydroxy oxynitrilase

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). 

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. 

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... 

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. 

In these synthesis, the optically active (R)-cyanohydrin is transformed into the corresponding a-hydroxy carboxylic ester and the hydroxyl funchon is achvated by sulfonylahon. The treatment of the corresponding intermediate with tetra-hydrothieno[3,2-c]pyridine stereoselectively yields the (S)-configured clopidogrel (Scheme 10.23). In the second case, a mutant of the recombinant almond (Pmnus amigdalus) (R)-oxynitrilase isoenzyme 5 catalyzes the formation of enantiopure (R)-2-hydroxy-4-phenylbutyronitrile [54]. Reaction of the sulfonylated hydroxyester derivative with the corresponding dipeptide leads to the formation of enalapril or lisinopril (Scheme 10.24). 

IS)-hydroxy(3-phenoxyphenyl)acetonitrile by hydrocyanation with oxynitrilase... 

In basic chemicals, nitrile hydratase and nitrilases have been most successful. Acrylamide from acrylonitrile is now a 30 000 tpy process. In a product tree starting from the addition of HCN to butadiene, nicotinamide (from 3-cyanopyridine, for animal feed), 5-cyanovaleramide (from adiponitrile, for herbicide precursor), and 4-cyanopentanoic acid (from 2-methylglutaronitrile, for l,5-dimethyl-2-piperidone solvent) have been developed. Both the enantioselective addition of HCN to aldehydes with oxynitrilase and the dihydroxylation of substituted benzenes with toluene (or naphthalene) dioxygenase, which are far superior to chemical routes, open up pathways to amino and hydroxy acids, amino alcohols, and diamines in the first case and alkaloids, prostaglandins, and carbohydrate derivatives in the second case. 

Oxynitrilase (Hydroxy Nitrile Lyase, HNL) Cyanohydrins from Aldehydes... 

Figure 7.8 a) Enzymatic (oxynitrilase-catalyzed) formation of cyanohydrins from aldehydes, b) Chemical hydrolysis of cyanohydrins to hydroxy acids. 

The hydroxynitrile lyase (HNL) class of enzymes, also referred to as oxynitrilases, consists of enzymes that catalyze the formation of chiral cyanohydrins by the stereospecific addition of hydrogen cyanide (HCN) to aldehydes and ketones (Scheme 19.36).275 279 These chiral cyanohydrins are versatile synthons, which can be further modified to prepare chiral a-hydroxy acids, a-hydroxy aldehydes and ketones, acyloins, vicinal diols, ethanolamines, and a- and P-amino acids, to name a few.280 Both (R)- and (.S )-selective HNLs have been isolated, usually from plant sources, where their natural substrates play a role in defense mechanisms of the plant through the release of HCN. In addition to there being HNLs with different stereo-preferences, two different classifications have been defined, based on whether the HNL contains a flavin adenine dinucleotide (FAD) co-factor. 

Fig. 6 A possible route to 3,3,3-trifluoro-2-hydroxy-2-methyl-propionic acid using an oxynitrilase. The route to the (S)-cya-nohydrin and (S)-acid is shown.

About 3000 plant species are known to release HCN from their tissues, a process which is known as cyanogenesis[17, 181. Storage compounds are cyanohydrins where the hydroxy function is glycosylated to a carbohydrate or protected as a fatty acid ester. The plant defence mechanism in the case of sugar compounds is a two-step reaction. Initially a glycosidase liberates the cyanohydrin moiety, which is cleaved either spontaneously by base catalysis or enzymatically by the action of oxynitrilases to release the corresponding carbonyl compound and HCN1191. 

Cyanohydrin formation Aldehyde or ketone 2-Hydroxy nitrile Synthesis of 2-hydroxy nitriles Oxynitrilase (Hydroxynitrile lyase)... 

The NHase and amidase were largely nonselective for cyanohydrins and the corresponding 2-hydroxy amides, respectively, but they were suitable for the hydrolysis of enantiopure cyanohydrins prepared from aldehydes and HCN by oxynitrilases [89, 90]. The cascade of NHase and amidase, in which the latter enzyme catalyzed an acyl transfer reaction, was suitable for the preparation of... 

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